Valveless fluidic switching flowchip and uses thereof

ABSTRACT

Provided are valveless microfluidic flowchips comprising fluid flow barrier structures or configurations. Further provided are systems and methods having increased fluid transfer control in a valveless microfluidic flowchip. The systems and methods can be used in the present valveless microfluidic flowchips as well as in currently available valveless microfluidic flowchips.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with government support under Contract No.EP-D-15-007 awarded by the United States Environmental ProtectionAgency. The government has certain rights in the invention.

INCORPORATION BY REFERENCE

An Application Data Sheet is filed concurrently with this specificationas part of the present application. Each application that the presentapplication claims benefit of or priority to as identified in theconcurrently filed Application Data Sheet is incorporated by referenceherein in its entirety and for all purposes.

STATEMENT OF JOINT RESEARCH AGREEMENT

The subject matter and the claimed invention were made by or on behalfof HJ Science & Technology, Inc. of Berkeley, CA and Protein Fluidics,Inc. of Burlingame, CA, under a joint research agreement titled“DEVELOPMENT AGREEMENT between HJ SCIENCE & TECHNOLOGY, INC. and PROTEINFLUIDICS, INC.” The subject matter disclosed was developed and theclaimed invention was made by, or on behalf of, one or more parties tothe joint research agreement that was in effect on or before theeffective filing date of the claimed invention, and the claimedinvention was made as a result of activities undertaken within the scopeof the joint research agreement.

BACKGROUND

Reconfigurable microfluidic systems based on networks of hydrophobicchannels using valve-less fluidic switching can be used for multipleapplications. Challenges are encountered with implementation of thistechnology due to robustness of the hydrophobic barriers and therequirement of various fluid transfer events.

Currently known reconfigurable microfluidic systems utilize hydrophobicbarriers (HPB) between connected wells and channels to control fluidmovement. The devices use straight channels connected to wells, andprocesses for fluid control that implement three pressures: High, Low,and Vacuum, where the low pressure is nominally atmospheric pressure,the high gas pressure moves fluid from a source well, through aconnecting channel, to a destination well, and the destination well iskept at low pressure (atmosphere) during this transfer. At the end of apressure cycle step to move fluid from a source well to a destinationwell, the connecting channel has been emptied to reestablish thehydrophobic barrier between the source well and channel.

SUMMARY

In one aspect, provided is a valveless microfluidic flowchip. In someembodiments, the flowchip comprises one or more networks of microfluidiccavities connected by microfluidic channels, wherein reservoirs arecavities that are connected to only one channel each, and nodes arecavities that are connected to two or more channels each; wherein: i) afirst plurality of the channels connect only two cavities each; ii) asecond plurality of the channels comprise one or more fluid flow barrierstructures or configurations; and iii) a plurality of the cavitiesinclude a gas pressure port. In some embodiments, the first and secondpluralities of the channels can be the same, different, or partially thesame (e.g., overlapping). In some embodiments, the one or more fluidflow barrier structures or configurations are located at or near aninterface of the cavity with the channel. In some embodiments, the oneor more fluid flow barrier structures or configurations increase channelresistance to fluid flow or the pressure required to move fluid by atleast about 20%, e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, ormore, e.g., in comparison to a channel that does not have a fluid flowbarrier structure or configuration. In some embodiments, one or more ofthe microfluidic channels are hydrophobic or comprise a hydrophobiccoating. In some embodiments, the one or more fluid flow barrierstructures or configurations comprise a constriction or narrowing of thechannel, ribs, and/or a non-linear path. In some embodiments, the one ormore fluid flow barrier structures or configurations comprise a geometryselected from the group consisting of serpentine or S-curve geometry, ajunction, a fishbone or a split channel. In some embodiments, the one ormore fluid flow barrier structures or configurations comprise a void(e.g., a sealed cavity) located in-line with the channel. In someembodiments, one or more or a plurality of the cavities are notcylindrical and comprise a concave curvature at the junction of thecavity with one or more channels, such that the cavity forms peninsulasthat extend from the cavity towards one or more channels (e.g., thecavity is in the shape of a lilypad). In some embodiments, one or moreor a plurality of the cavities comprises a perpendicular entrance of oneor more channels into the cavity, such that there is a sharp (e.g., ofabout 90°, e.g., not gradual or flared) change in geometry where thechannel enters the cavity. In some embodiments, the nodes are configuredsuch that entrance (e.g., input, transfer) channel and exit (e.g.,output, assay) channel junctions are located in different verticalplanes, e.g., where the input channel enters at the side of the node andthe output channel exits from the center of the node. In someembodiments, a region is created between the entrance and exit channelsthat can retain a defined amount of fluid when a cavity is emptiedduring a transfer process. In some embodiments, the flowchip comprises ahydrophobic fluidic layer (115) comprised of one or more polymersselected from the group consisting of polypropylene (PP), a cyclicolefin polymer (COP), a cyclic olefin copolymer (COC); a fluoropolymersuch as polytetrafluoroethylene (PTFE), fluorinated ethylene propylene(FEP, a copolymer of hexafluoropropylene and tetrafluoroethylene),perfluoro alkoxy polymer resin (PFA); and a silicone polymer such aspolydimethylsiloxane (PDMS). In some embodiments, the polymers can bemodified to increase their hydrophobicity through use of additives,surface coatings, or surface modifications. In some embodiments, one ormore or a plurality of the cavities can be connected with up to 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 channels each. In someembodiments, each network in the one or more networks comprises aninput/output channel, the input/output channel having a greaterresistance to fluid flow than that of the microfluidic channels. In someembodiments, each flowchip can contain a plurality of networks spaced atregular intervals with the number, spacing and density of networksdefined by industry standards such as American National StandardsInstitute (ANSI) Society for Laboratory Automation and Screening (SLAS)4-2004 (R2012).

In a further aspect, provided are valveless microfluidic systems. Insome embodiments, the systems comprise a flowchip as described above andherein, wherein the system comprises a pressure sequencer including aset of gas valves, the pressure sequencer connected by pneumaticdelivery channels to: (1) a high gas pressure gas source; (2) anintermediate gas pressure gas source; (3) a low pressure gas source; andoptionally, (4) a partial vacuum pressure gas source; and to at leastone cavity in the flow chip. In some embodiments, the systems comprise:a) a flowchip comprising: one or more networks of microfluidic cavitiesconnected by microfluidic channels, wherein: reservoirs are cavitiesthat are connected to only one channel each, and nodes are cavities thatare connected to two or more channels each, wherein: i) a firstplurality of the channels connect only two cavities each; ii) a secondplurality of the channels have a greater resistance to fluid flow thanthat of the nodes; and iii) a plurality of the cavities include a gaspressure port; and b) a pressure sequencer comprising a set of gasvalves, the pressure sequencer connected by pneumatic delivery channelsto: (1) a high gas pressure gas source; (2) an intermediate gas pressuregas source; (3) a low pressure gas source; and optionally, (4) a partialvacuum pressure gas source; and to at least one cavity within theflowchip. In some embodiments, the first and second pluralities of thechannels can be the same, different, or partially the same (e.g.,overlapping). In some embodiments, the pressure sequencer is configuredto apply a high gas pressure, an intermediate gas pressure, a low gaspressure, and optionally, a partial vacuum pressure to the at least onecavity according to pressure sequence data, where the high gas pressureis greater than the intermediate gas pressure, the intermediate gaspressure is greater than the low gas pressure, and the low gas pressureis greater than the partial vacuum gas pressure, and the partial vacuumpressure is less than atmospheric pressure. In some embodiments, thepressure sequencer is configured to concurrently apply a combination ofgas pressure and partial vacuum to at least one cavity. In someembodiments, the second plurality of the channels comprises one or morefluid flow barrier structures or configurations. In some embodiments,the one or more fluid flow barrier structures or configurations arelocated at or near an interface of the cavity with the channel. In someembodiments, the one or more fluid flow barrier structures orconfigurations increase channel resistance to fluid flow or the pressurerequired to move fluid by at least 20%, e.g., at least about 25%, 30%,35%, 40%, 45%, 50%, or more, in comparison to a channel that does nothave a fluid flow barrier structure or configuration. In someembodiments, one or more of the microfluidic channels are hydrophobic orcomprise a hydrophobic coating. In some embodiments, the one or morefluid flow barrier structures or configurations comprise a constrictionor narrowing of the channel, ribs and/or a non-linear path. In someembodiments, the one or more fluid flow barrier structures orconfigurations comprises a geometry selected from the group consistingof serpentine or S-curve geometry, a junction, a fishbone or a splitchannel. In some embodiments, the one or more fluid flow barrierstructures or configurations comprise a void (e.g., a sealed cavity)located in-line with the channel. In some embodiments, one or more or aplurality of the cavities comprises a perpendicular entrance of one ormore channels into the cavity, such that there is a sharp (e.g., ofabout 90°, e.g., not gradual or flared) change in geometry where thechannel enters the cavity. In some embodiments, the nodes are configuredsuch that entrance (e.g., input, transfer) channel and exit (e.g.,output, assay) channel junctions are located in different verticalplanes, e.g., where the input channel enters at the side of the node andthe output channel exits from the center of the node. In someembodiments, a region is created between the entrance and exit channelsthat can retain a defined amount of fluid when a cavity is emptiedduring a transfer process.

In a related aspect, provided is a system for moving a quantity ofliquid from a source cavity to a destination cavity in a network ofmicrofluidic cavities, wherein the source cavity and the destinationcavity are separated by a valveless microfluidic channel having aresistance to fluid flow greater than that of the source cavity, themethod comprising: (i) a receptacle for receiving and engaging with aflowchip comprising the network of microfluidic cavities; (ii) apressure sequencer comprising a set of gas valves and configured to beconnected to a first gas source for producing a high gas pressure inmicrofluidic cavities, a second gas source for producing a low pressurein microfluidic cavities, and a third gas source for producing anintermediate gas pressure in microfluidic cavities, and optionally, afourth partial vacuum source wherein the high gas pressure is greaterthan the low pressure, the intermediate gas pressure is less than thehigh gas pressure but greater than the low pressure, and theintermediate gas pressure is insufficiently great overcome resistance tofluid flow in the microfluidic channel when the source cavity issubstantially empty of the liquid, and the partial vacuum is less thanatmospheric pressure, wherein the pressure sequencer can apply anypressure state to any cavity within the flowchip; and (iii) a controllerconfigured to direct the pressure sequencer to: (a) apply the high gaspressure to the source cavity and to all other cavities connected to thesource cavity excepting the destination cavity, while applying the lowpressure to the destination cavity, to move a portion of the quantity ofliquid from the source cavity, through the microfluidic channel, and tothe destination cavity, and (b) apply an intermediate gas pressure tothe source cavity before the quantity of liquid is completely removedfrom the source cavity, wherein the intermediate gas pressure issufficiently great to push at least some of the quantity of liquidremaining after (a) to the destination cavity, but avoids introducinggas into the microfluidic channel. In some embodiments, the methodcomprises further applying partial vacuum to a destination cavity orother connecting cavity, the partial vacuum being applied for a timesufficient to evacuate fluid from the destination cavity. In someembodiments, a defined amount of fluid remains in the source cavity in aregion between the entrance and exit channels.

In some embodiments of the systems, the pressure sequencer is configuredto apply a one or more pressure modes selected from the group consistingof constant pressure, pulsing pressures, increased ramping pressures anddecreased ramping pressures. In some embodiments, the pressure sequenceris configured to apply pulsing pressures and a pulse width modulation(PWM) with a duty factor in the range of from about 1% to about 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In some embodiments, thepressure sequencer is configured to apply increased and/or decreasedramping pressures comprising rise and/or fall times in the range ofabout 10 msec to about 20 msec, 50 msec, 100 msec, 250 msec, 500 msec,750 msec or 1 sec. In some embodiments, one or more of the microfluidicchannels are hydrophobic or comprise a hydrophobic coating. In someembodiments, the system comprises a flowchip as described above andherein. In some embodiments, i) the high gas pressure is in the range ofabout 5 kPa to about 10 kPa, 20 kPa, 30 kPa, 40 kPa, 50 kPa, 60 kPa, 70kPa, 80 kPa, 90 kPa or 100 kPa, e.g., in the range of about 10 kPa toabout 60 kPa; and/or ii) the intermediate gas pressure is in the rangeof about 0.5 kPa to about 1 kPa, 2 kPa, 3 kPa, 4 kPa, 5 kPa, 6 kPa, 7kPa, 8 kPa, 9 kPa or 10 kPa; and/or iii) the optional partial vacuumpressure is in the range of about −5 kPa to about −10 kPa, −20 kPa, −30kPa, −40 kPa, −50 kPa, −60 kPa, −70 kPa, −80 kPa, −90 kPa, or −100 kPa.Generally, the high gas pressure is greater than the intermediate gaspressure, the intermediate gas pressure is greater than the low gaspressure, and the low gas pressure is greater than the partial vacuumgas pressure, and the partial vacuum pressure is less than atmosphericpressure. In some embodiments, fluid flow rate under high gas pressurethrough the first plurality of microfluidic channels is from about 0.1μL/second to about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0,3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10.0 μL/second. In someembodiments, fluid flow rate under intermediate gas pressure through thefirst plurality of microfluidic channels is from about 0.01 μL/second toabout 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0μL/second. Generally, the fluid flow rate under high gas pressure isfaster than the fluid flow rate under intermediate gas pressure. In someembodiments, a plurality of the microfluidic channels present ahydrophobic pressure barrier to fluid flow that is less than thepressure difference between the high gas pressure and the low gaspressure. In some embodiments, the pressure sequencer is configured toapply or follow a fluid transfer rule in which: (1) high gas pressure isapplied to an origin or source cavity from which a fluid is transferredand low gas pressure is applied to a destination cavity to which thefluid is transferred, the high gas pressure being applied for a timet(1) sufficient to overcome hydrophobic and/or hydrostatic barriers andstart fluid flowing from the origin or source cavity into a microfluidicchannel connecting the origin or source cavity to the destinationcavity; (2) intermediate gas pressure is applied to the origin or sourcecavity and low pressure is applied to the destination cavity such thatfluid continues to move through the connecting channel, the intermediategas pressure being applied for a time t(2) sufficient to empty theorigin or source cavity of fluid but of a pressure insufficient to expelfluid out of the channel; whereby the origin or source cavity is emptiedof fluid and the fluid is moved into the channel and destination cavity.In some embodiments, a defined amount of fluid remains in the sourcecavity in a region between the entrance and exit channels. In someembodiments, the pressure sequencer is configured to follow a fluidtransfer rule further in which: (3) partial vacuum is applied to thedestination channel while low pressure is applied to the source cavity210 such that fluid is evacuated or removed from the destination cavity220 through the gas port. In some embodiments, the pressure sequencer isconfigured to concurrently apply a combination of gas pressure andpartial vacuum to at least one cavity. In some embodiments, partialvacuum is applied to the destination cavity 220 through a port orchannel in fluid communication with the bottom surface of thedestination cavity 230 and fluid is evacuated or removed from the bottomsurface of the destination cavity. See, e.g., FIGS. 5 and 6 . In someembodiments, gas pressure is applied to the destination cavity 220through a port or channel in fluid communication with the top opening ofthe destination cavity 240 (e.g., above or over the meniscus of thefluid in the destination cavity) concurrently with partial vacuum beingapplied to the destination cavity through a port or channel in fluidcommunication with the bottom surface of the destination cavity 230(e.g., below or under the fluid in the destination cavity). In someembodiments, time t(1) is for a time period that is stopped or endedbefore the quantity of liquid is completely removed from the sourcecavity, e.g., a time period sufficient to drain at least about 10% andup to about 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the fluid volumefrom the origin or source cavity. In some embodiments, the pressuresequencer is further connected to a very high gas pressure source, andthe pressure sequencer is configured to apply a very high gas pressure,wherein the very high gas pressure is greater than the high gaspressure. In some embodiments, the very high gas pressure is at leastabout 100 kPa, e.g., at least about 125 kPa, 150 kPa, 175 kPa, 200 kPa,or higher. In some embodiments, the pressure sequencer is configured toapply or follow a fluid transfer rule in which the partial vacuum gaspressure is applied to a destination cavity to which a fluid is drawnvia its input/output channel and low gas pressure is applied to anyother cavity connected to the destination cavity by a channel. In someembodiments, one or more networks comprise j rows and k columns ofcavities, j and k being positive integers, cavities in each row orcolumn being connected in series.

In a further aspect, provided are methods for arranging fluid in amicrowell plate. In some embodiments, the methods comprise operating thevalveless microfluidic system as described above and herein according toa set of pressure sequence data that causes the fluid to be drawn intothe system from an origin or source cavity of the microwell plate andexpelled into a destination cavity of the microwell plate, wherein airis not introduced into a microfluidic channel downstream of an origin orsource cavity.

In a further aspect, provided are methods for performing a homogenousassay with j samples and k reagents. In some embodiments, the methodscomprise operating the valveless microfluidic system as described aboveand herein, with pressure sequence data that causes each of the jsamples to be exposed to the k reagents thereby producing j outputsolutions, wherein air is not introduced into a microfluidic channeldownstream of an origin or source cavity.

In a further aspect, provided are methods for performing a multiplexedimmunoassay. In some embodiments, the methods comprise operating thevalveless microfluidic system as described above and herein, wherein thesystem comprises two or more networks, the system operated according topressure sequence data such that the pressure sequencer directs fluidflows in the system that cause different kinds ofsample-analyte-capture-analyte reactions to occur in different networks,but the same kind of detection reagent reaction to occur in a pluralityof networks, wherein air is not introduced into a microfluidic channeldownstream of an origin or source cavity. In some embodiments, theimmunoassay fluid comprises a buffer having a pH in the range of 6-11,e.g., pH in the range of 6-9, e.g., a pH in the range of about 7-9 or apH in the range of 9-11, one or more blocking agents or proteinsolutions and one or more surfactants. In specific embodiments, theimmunoassay fluid comprises phosphate buffered saline (PBS),tris-buffered saline (TBS) or a bicarbonate buffer, albumin (e.g.,bovine serum albumin (BSA)), Tween-20, Triton-X, or other surfactantsand optionally glycerol.

In a further aspect, provided are methods of moving a quantity of liquidfrom a source cavity to a destination cavity in a network ofmicrofluidic cavities. In some embodiments, the methods are executedusing a valveless microfluidic flowchip having a source cavity and adestination cavity separated by a valveless microfluidic channel havinga resistance to fluid flow greater than that of the source cavity. Insome embodiments, the methods comprise: (a) applying a high gas pressureto the source cavity, and all other cavities connected to the sourcecavity excepting the destination cavity, while applying a low pressureto the destination cavity to move a portion of the quantity of liquidfrom the source cavity, through the microfluidic channel, and to thedestination cavity, wherein the high gas pressure is greater than thelow pressure; and (b) applying an intermediate gas pressure to thesource cavity before the quantity of liquid is completely removed fromthe source cavity, wherein the intermediate gas pressure is lower thanthe high gas pressure but higher than low pressure, and wherein theintermediate gas pressure is sufficiently great to push at least some ofthe quantity of liquid remaining after (a) to the destination cavity,but insufficiently great overcome resistance to fluid flow in themicrofluidic channel, and thereby avoid introducing gas into themicrofluidic channel. In some embodiments, the pressure sequencer isconfigured to follow a fluid transfer rule further in which partialvacuum is applied to the destination channel while low pressure isapplied to the source cavity such that fluid is evacuated or removedfrom the destination cavity through the gas port. In some embodiments,partial vacuum is applied to the destination cavity 220 through a portor channel in fluid communication with the bottom surface of thedestination cavity 230 and fluid is evacuated or removed through thebottom surface of the destination cavity. In some embodiments, gaspressure is applied to the destination cavity 220 through a port orchannel in fluid communication with the top opening of the destinationcavity 240 (e.g., above or over the meniscus of the fluid in thedestination cavity) concurrently with partial vacuum being applied tothe destination cavity through a port or channel in fluid communicationwith the bottom surface of the destination cavity 230 (e.g., below orunder the fluid in the destination cavity). In some embodiments, the oneor more of the microfluidic channels are hydrophobic or comprise ahydrophobic coating. In some embodiments, the intermediate gas pressureis insufficiently great to introduce gas into the microfluidic channeleven when all of the quantity of liquid has been removed from the sourcecavity. In some embodiments, less than about 90% of the liquid isremoved from the source cavity before applying the intermediate gaspressure. In some embodiments, the method is performed using a system asdescribed above and herein.

In a further aspect, methods of performing assays using cells orcellular structures are providing. The methods may involve providing amicrofluidic flowchip comprising one or more networks of microfluidiccavities connected by microfluidic channels, wherein nodes are cavitiesthat are connected to two or more channels each, wherein at least onenode comprises a first junction with an input channel and a secondjunction with an output channel, wherein the first junction and thesecond junction are located at different vertical planes, and whereinthe node includes a main region and a defined region having a definedvolume, the defined region disposed below the main region; and directingcells or cellular structures from the main region to the defined region.In some embodiments, directing cells or cellular structures involves tothe defined region comprises flowing fluid from the input channelthrough the defined region to the output channel. In some embodiments,directing cells or cellular structures comprises to the defined regionincludes introducing fluid from the input channel into the main regionat an angle. In some embodiments, the method is performed using aflowchip or system as described above and herein.

These and other aspects are described below with reference to thedrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. A. An example flowchip depicting 4 microfluidic networks.B. An illustrative configuration of two cavities and emanatingmicrofluidic channels that do not have any fluid flow barrier structuresor configurations.

FIG. 2 illustrates a valveless microfluidic flowchip, seen in crosssection. The fluid state depicted in this cross-sectional view isidentical to FIG. 3A.

FIGS. 3A-3F illustrate a schematic of an implementation for transferringfluid from a Source well (e.g., well A) to a Destination well (e.g.,well B) through a connecting channel. HP=high pressure; IP=intermediatepressure; LP=low pressure.

FIGS. 4A-4F illustrate a schematic of an implementation for transferringfluid from a Source well (e.g., well B) to a Destination well (e.g.,well C) through a connecting channel and then evacuating the fluid fromthe Destination well. HP=high pressure; IP=intermediate pressure; LP=lowpressure, VAC=partial vacuum.

FIG. 5 illustrates a valveless microfluidic flowchip, seen in crosssection with a second manifold interfaced to the bottom of the flowchip.The fluid state depicted in this cross-sectional view is identical toFIG. 6D.

FIGS. 6A-6F illustrate a schematic of an implementation for transferringfluid from a Source well (e.g., well B) to a Destination well (e.g.,well C) through a connecting channel and then evacuating the fluid fromthe Destination well where the evacuation port is separate from thepressure port. HP=high pressure; IP=intermediate pressure; LP=lowpressure, VAC=partial vacuum.

FIGS. 7A-7C illustrate different fluid flow barrier structures orconfigurations.

FIGS. 8A-8C illustrates the results of flowrates for an assay bufferthat were measured for the three structures at different appliedpressures.

FIGS. 9A-9B illustrates a void feature (e.g., fluid flow barrierstructure or configuration) that is used to increase hydrophobic barrierand hydrostatic resistance in channels. The Void diameters (ϕ)) for anabout 50 μm wide (w) channel range from about 100 to about 1500 μm. TheVoid heights (h) for an about 50 μm high channel range from about 50 toabout 500 μm. The optimum diameter and height ranges are dependent onthe input channel geometry. The Void cross sections can be circular orelliptical. The Void walls can be perpendicular to the channels or haveslight angles (e.g., about 0 to about 20 degrees) to facilitatefabrication.

FIG. 10 illustrates the results of breakthrough pressures for voidfeatures of different heights.

FIG. 11A-11B illustrate a rib feature that is used to increasehydrophobic barrier and hydrostatic resistance in channels. The Ribheight constriction (Δh) for an about 50 μm high channel ranges fromabout 5 to about 40 μm. The length of the Rib constriction (L) for anabout 50 μm wide (w) channel can range from about 100 to about 1000 μm.Height and length ranges are dependent on the input channel geometry.

FIG. 12 illustrates the results of breakthrough pressures for ribfeatures of different heights.

FIG. 13 illustrates the relationship between breakthrough pressure andcalculated capillary pressure for rib features of different dimensions.

FIGS. 14A-14B illustrate two designs for junctions between channels andcavities. Depicted are the molds around which a cavity-channel junctionis formed. A. A “Landing Pad” gap exists between the channel and cavitycausing a more gradual change of geometry between the channel and cavityand providing a microcapillary connection to other channel junctions. Inthis version, when the pin is removed, the “landing pad” mold leaves a“lip” where the bottom surface diameter is wider than the walls of thecavity. Further, the channel is flared at the junction with the cavity.B. No gap exists in the plane of the channel and the channel entersstraight into the cavity. In this version, the mold does not leave anylip, and the bottom surface diameter is equal to the walls of thecavity. Moreover, there is a sharp change in geometry between thechannel and cavity, because the junction of the channel with the cavityis perpendicular. No microcapillary connection exists to other channeljunctions.

FIGS. 15A-15C illustrate a design for junctions between channels andcavities that includes multiple sharp angles for junctions of channelsthat transfer fluid into a cavity and a vertically isolated junction fora channel that transfers fluid out of a cavity (e.g., into an assaychannel). FIG. 15A shows a top view of a cavity with such junctionswhere the sharp edges (e.g., substantially perpendicular relative to thetransfer channel, e.g., a sharp corner that is not flared or rounded) ofan input (transfer) channel can be seen. FIG. 15B shows a bottom view ofa cavity with multiple input (transfer) channels and a single output(assay) channel. FIG. 15C shows a cross section of a channel through aninput (Transfer) and output (Assay) channel. The input junctions arelocated in a different vertical plane than the output junction providingenhanced isolation of the junctions. For example, one or more inputchannels can enter at or near the outer diameter of the node and anoutput channel can exit at or near the center of the node, e.g., asdepicted in FIGS. 15A-15B. Fluid can also be transferred out of a cavitythrough an entrance port such that a defined amount of fluid remains inthe cavity in a region between the entrance and exit ports.

FIG. 15C shows the defined region, which has a defined volume thatdetermines the defined amount of fluid that remains in the cavity whenfluid is transferred from the cavity to the transfer channel through theentrance port. It should be noted that while the junction between thetransfer channel and the cavity in FIG. 15C is referred to an “entranceport” and the junction between the cavity and the assay channel isreferred to as an “exit port” in this description, fluid may betransferred into or out of the cavity in any direction. In someembodiments, the dimensions of a junction may be different than that ofthe main part of the channel. In some embodiments, the dimensions of thea junction are smaller than the main part of the channel. This canfurther reduce leakage.

In some embodiments, a device including a defined region as shown inFIG. 15C may be used for assays that use cells or cellular structuressuch as spheroids, microtissues, islets, and organoids. The cells orcellular structures may be directed from the main portion of the cavityinto the defined region, which is filled with a defined amount of fluid.This prevents the cells or cellular structures from being located on thesides of the main cavity region, for example, where they may be driedout when the main cavity is emptied of fluid. To direct the cells orcellular structures into the defined region, in some embodiments, afluid may be introduced into one or more entrance ports (as shown inFIGS. 15A-C) at the top of the defined region and out of the exit port(into the assay channel of FIGS. 15A-C). This creates a fluid flow paththat directs any cells or cellular structures that are in the maincavity into the defined region. The cavity volume that is above of thedefined region may be described as the main region of the cavity.

In some embodiments, the entrance ports may introduce fluid into thecavity at an angle. For example, the vertical channel shown in FIG. 15Cthat connects the transfer channel to the cavity may be angled in adirection into the page. Fluid introduced into the cavity then cancreate a vortex that would “swirl” the cells or cellular structures tothe center of the cavity and into the defined region.

FIGS. 16A-16B illustrate the improvement in fluid control provided bythe geometrical features shown in FIG. 15A-15C. A fluid with highsurfactant concentration and a fluorescent dye (fluorescein) is loadedinto the wells and then after a period of 60 min the wells and channelsare imaged with a fluorescence microscope (4× objective, 490 nmexcitation, 530 nm emission). FIG. 16A shows results from a device withfeatures shown in FIG. 14B. Significant passive leakage into thechannels is seen with a native COC surface. The addition of surfacecoating that enhances the hydrophobic barrier reduces passive leakage.FIG. 16B shows results from a device with features shown in FIG. 15 . Nopassive leakage is observed with native COC surface indicating a higherbarrier to fluid movement.

FIGS. 17A-17B illustrates the reagent loading configuration forperforming a flowchip ELISA. The assay protocol is divided into a 1^(st)Half (FIG. 17A) and a 2^(nd) Half (FIG. 17B). Reagent locations areindicated on cross-sectional views of the flowchip shown in FIG. 1A.Well numbers are indicated below the flowchips.

FIG. 18 shows the Standard Response Curves for MCP-1, IL-8, and IL-6generated from the flowchip ELISA system. The linear fit parametersshown in the figure were used to quantify the amount of the cytokinespresent in cell supernatants for the multiparametric inflammation assay.

FIG. 19 shows the upregulation of MCP-1, IL-8, and IL-6 in HUVECs by aninflammatory cytokine mixture of TNF-α, IL-1β, and IFN-γ after about 20hours at 37° C. The maximum concentrations of the compounds (relativevalue=100) were about 5 ng/well, about 1 ng/well, and about 100 ng/wellrespectively.

FIGS. 20A-20D shows the concentration dependent effect ofanti-inflammatory compounds SB202190, MG-132, and AG-126 on HUVECsstimulated with an inflammatory cytokine mixture of TNF-α, IL-1β, andIFN-γ for 20 hours at 37° C. HUVEC inflammation response as shown byupregulation of IL-8 (FIG. 19A), IL-6 (FIG. 19B), and MCP-1 (FIG. 19C)was clearly diminished by all three compounds. Each curve was fit with a4-Parameter function and the corresponding EC₅₀ values are shown in FIG.19D.

FIGS. 21A-21B illustrate a second example flowchip with improved fluidcontrol features. A. An example flowchip depicting 4 microfluidicnetworks. B. A zoomed region of one microfluidic network showing thelocation of fluid flow barrier structure (voids and ribs) that wereadded to improve fluid control.

FIG. 22 illustrates the reagent loading configuration for performing aflowchip ELISA in the improved flowchip shown in FIG. 21 . The assayprotocol is performed with a single reagent loading step. Reagentlocations are indicated on cross-sectional views of the flowchip. Wellnumbers are indicated below the flowchips.

FIGS. 23A-23B illustrate the improvement in assay performance realizedby the flowchip device shown in FIG. 21 . FIG. 23A shows standardresponse curves for an IL-6 ELISA from the device shown in FIG. 1 (FC-1)and device shown in FIG. 21 (FC-2) with improved fluid control. Assayperformance metrics are given in FIG. 23B showing significant assayimprovement using a device with enhanced fluid control features.

FIGS. 24A-24B show the upregulation of IL-8 (FIG. 24A) and IL-1b (FIG.24B) in THP-1 cells after stimulation with different concentrations ofLPS after about 20 hours at 37° C.

FIGS. 25A-25B show the concentration dependent effect ofanti-inflammatory compounds SB202190, Moxifoxacin, and PDTC on THP-1cells stimulated with PMA and LPS for 20 hours at 37° C. THP-1 cellularinflammation response as shown by upregulation of IL-8 (FIG. 25A) wasclearly diminished by all three compounds. Each curve was fit with a4-Parameter function and the corresponding EC₅₀ values are shown in FIG.25B.

DETAILED DESCRIPTION

1. Introduction

The flowchips, systems and methods described herein address thechallenges presented by the currently available reconfigurablemicrofluidic systems in that the high gas pressure needs to exert enoughforce to overcome the initial hydrostatic and hydrophobic barriers tomove fluid through the channel, but insufficient force to force airthrough the destination well once the channel empties. The flowchips,systems and methods are based, in part, on the discovery and utilizationof a narrow process window to achieve this balance, which can beadjusted by adjusting parameters, such as channel dimensions, flowchipmaterial, and fluid composition. The present flowchips and systems aresuitable for running multi-step assays (e.g., such as ELISAs), which canrequire flowchips with multiple channels of varying cross-sectionalareas and lengths, and involve reagents with different physicalcharacteristics (e.g., buffers, substrates, stopping solutions, blockingagents, etc).

Herein we describe methods for control of fluid movement in valvelessmicrofluidic flowchips that use high, intermediate, low and vacuumpressure settings and surface tension induced fluid resistance at thewell/channel interface (WCI) to improve robustness. Additionally, weprovide a system and method for removing fluid from a flowchip usingpartial vacuum. Further, we provide flowchips with channels havingstructural fluid flow barrier structures or configurations that increasethe hydrophobic and hydrostatic barriers without substantiallycompromising overall fluid flow.

2. Valveless Microfluidic Flowchips

Provided are valveless (e.g., capillary force driven) microfluidicflowchips. In some embodiments, the flowchips comprise one or morenetworks of microfluidic cavities connected by microfluidic channels,wherein reservoirs are cavities that are connected to only one channeleach, and nodes are cavities that are connected to two or more channelseach; wherein: i) a first plurality of the channels connect only twocavities each; ii) a second plurality of the channels comprise a fluidflow barrier structure or configuration; and iii) a plurality of thecavities include a gas pressure port. In some embodiments, the first andsecond pluralities of the channels can be the same, different, orpartially the same (e.g., overlapping). A “fluid flow barrier structureor configuration” refers to a structural feature of a microfluidicchannel having increased fluid flow resistance. The pressure required topush fluid through a channel from a source well to a destination well isreferred to as the “breakthrough pressure”. Often, the structuralfeature is a highly non-linear deviation from a straight path betweenadjacent cavities, a narrowing or constriction in the channel (whetherstraight or otherwise), a void (e.g., a sealed cavity) in the channelthat introduces an abrupt and substantial change in geometry, includingan increase in channel dimensions (height and width), and/or a variationin the channel's surface condition (e.g., a roughening). Increased fluidflow resistance can be due to one or more forces resisting flow,including without limitation, resistive forces resulting from staticfriction, surface energy, surface tension, fluid density, and/or fluidviscosity.

The presently described flowchips are improved over valvelessmicrofluidic flowchips described in the art, e.g., U.S. PatentPublication Nos. US2017/0021351, US2017/0021352 and US2017/0021353(issued as U.S. Pat. No. 9,733,239), hereby incorporated herein byreference in their entireties for all purposes, in that a plurality ofthe channels in the present flowchips comprise a fluid flow barrierstructure or configuration allowing for more precise control of fluidflow and their use with intermediate positive pressures avoidintroduction of air bubbles or air gaps into the channels.

The fluid flow barrier structure or configuration can be locatedanywhere along the length of a microfluidic channel. In someembodiments, a fluid flow barrier structure or configuration is locatedat or near an interface of the cavity with the channel. In someembodiments, a fluid flow barrier structure or configuration is locatedessentially at the interface of a cavity and a microfluidic channel,e.g., at a distance in the range of from about 0 mm to about 5 mm, 6 mm,7 mm, 8 mm, 9 mm or 10 mm from a cavity. As appropriate, a microfluidicchannel can have one, two or more fluid flow barrier structures orconfigurations. In a microfluidic channel having two or more fluid flowbarrier structures or configurations, the fluid flow barrier structuresor configurations can be the same or different. The fluid flow barrierstructure or configuration can also incorporate an enhanced hydrophobicbarrier.

In some embodiments, the fluid flow barrier structure or configurationincreases channel resistance to fluid flow or the pressure required tomove fluid by at least about 20%, e.g., at least about 25%, 30%, 35%,40%, 45%, 50%, or more, e.g., in comparison to a channel that does nothave a fluid flow barrier structure or configuration (e.g., a straight,unconstricted channel). A fluid flow barrier structure or configurationcan be any structural configuration of a microfluidic channel thatincreases the resistance of fluid flow, e.g., in comparison to a linearor substantially linear and substantially unconstricted microfluidicchannel without the fluid flow barrier structure or configuration, e.g.,in comparison to a linear microfluidic channel having constant and fullwidth and height dimensions. In some embodiments, the fluid flow barrierstructure or configuration comprises a constriction or narrowing of thechannel, a rib feature, and/or a channel having a markedly non-linearpath. In some embodiments, the non-linearity is characterized by anabrupt change in the direction of a channel, e.g., which can be from 45degrees to 135 degrees, e.g., over a length of 1 to 5 channel widths.The number of changes, or turns in direction, can be from 1 to 10 ormore in sequence. A fluid flow barrier structure or configuration thatis a constriction or narrowing of the channel is illustrated in FIG. 7B;a rib feature is illustrated in FIG. 11 . In some embodiments, the fluidflow barrier structure or configuration comprises a geometry selectedfrom the group consisting of serpentine or S-curve geometry, a junction,a fishbone or a split channel. A serpentine or S-curve fluid flowbarrier structure or configuration is illustrated in FIG. 7A. In someembodiments, the fluid flow barrier structure or configuration comprisesa void (e.g., a sealed cavity) located in-line with the channel. A voidfluid flow barrier structure or configuration introduces an abrupt andsubstantial change in geometry, including increases in height and widthdimensions, as illustrated in FIGS. 9A-9B. In some embodiments, one ormore or a plurality of the cavities are not cylindrical and comprise aconcave curvature at the junction of the cavity with one or morechannels, such that the cavity forms peninsulas that extend from thecavity towards one or more channels (e.g., the cavity is in the shape ofa lilypad). See, e.g., FIG. 7C. In some embodiments, a sealed cavity orvoid is incorporated into a channel, e.g., as depicted in FIGS. 9A-9B.In some embodiments, a region of reduced height (e.g., a rib feature) isincorporated into a channel, e.g., as depicted in FIG. 11 . In someembodiments, a channel can have multiple fluid flow barrier structuresor configurations, e.g., 2, 3, 4 or more fluid flow barriers.

In some embodiments, one or more or a plurality of the cavities includea straight and perpendicular entrance of one or more channels into thecavity, such that there is a sharp change in geometry (e.g., 90° wherethe channel enters the cavity, e.g., as depicted in FIGS. 14A-14B.Perpendicular includes the intersection of a straight channel regionwith a small segment of a curved surface, as shown in FIGS. 14A-14B. Insome embodiments, the nodes are configured such that entrance (input,transfer) channel and exit (output, assay) channel junctions are locatedin different vertical planes, e.g., as depicted in FIGS. 15A-15B. Forexample, the input (transfer) channels can enter at one or more entrancepoints of the space above the node and the output (assay) channels canexit at one or more exit points at the bottom of the node. The inputchannels are located at or near the outer diameter of the cavity (e.g.,within about 1 mm of the outer edge of a 3 mm diameter cavity, e.g.,within the outer ⅓ of the diameter of the cavity) while the outputchannel is located at or near the center of the cavity (e.g., withinabout 1 mm of the center, e.g., within the inner ⅓ of the diameter ofthe cavity). In some embodiments, a defined amount of fluid remains inthe source cavity in a region between the entrance and exit channels.

Similar to the microfluidic systems described in U.S. Patent PublicationNos. US2017/0021351, US2017/0021352 and US2017/0021353 (issued as U.S.Pat. No. 9,733,239), the valveless microfluidic flowchips describedherein are based on networks of microfluidic cavities connected bymicrofluidic channels, which can be hydrophobic. Each cavity can beclassified as either a reservoir or a node, and includes a pressure portvia which gas pressure may be applied. Sequences of gas pressures,applied to reservoirs and nodes according to fluid transfer rules,enable fluid to be moved from any reservoir to any other reservoir in asystem.

The valveless microfluidic flowchips can be designed from the basiccomponents of reservoirs, nodes and channels to perform many differentmicrofluidic tasks including, e.g., homogenous and inhomogeneous assaysand microwell plate interfacing. The systems are scalable to any numberof fluid inputs and outputs, and they can be used to manipulate verysmall fluid volumes necessary for multiplexing samples with analytes toperform multiple simultaneous assays.

A microfluidic cavity is an internal volume for accumulating fluid inthe microfluidic flowchips. A reservoir is a microfluidic cavity that isconnected to only one microfluidic channel. A node is a microfluidiccavity that is connected to more than one microfluidic channel. Finally,a channel is a microfluidic passageway between nodes or reservoirs. Eachchannel in the present valveless microfluidic system connects at leasttwo cavities. Contemplated are flowchip designs where there are channelintersections and fluid flow is controlled by differential resistance indifferent channels.

Nodes are designed to present lower resistance to fluid flow than arechannels. The fluid flow resistance of a cavity or channel is inverselyproportional to the square of its cross sectional area. Therefore thedifference in flow resistance between a channel and a reservoir, orbetween a channel and a node, may be engineered via different crosssectional areas.

Reservoirs store fluids; e.g., samples or reagents. Nodes, on the otherhand, can store a fluid initially and also can store other fluids duringa sequence of fluid transfer steps. Provisions for automated loadingfluid into, or unloading fluid from, a reservoir may be provided, with asmall plastic tube extending from a reservoir to a glass bottle or withan automated pipette station being examples.

The valveless microfluidic flowchips can be implemented in a variety ofways as long as: reservoirs, nodes, channels and pressure ports areprovided; and resistance to fluid flow is greater in the channels thanin the nodes. In some embodiments, the channels are hydrophobic, e.g.,to prevent fluid flow when pressures at the two ends of a channel areequal or nearly so. In the present flowchips, fluid flow throughmicrofluidic channels is controlled by gas pressure differences appliedto the cavities, e.g., reservoirs and nodes. Fluid flow through ahydrophobic channel exhibits a pronounced threshold effect. At first, nofluid flows as the pressure difference from one end of the channel tothe other is increased. However, once a threshold pressure difference isreached, fluid flow rate through the channel increases in proportion toapplied pressure difference. The hydrophobicity of channels sets thethreshold pressure difference, and the difference between high and lowpressures used in a system is designed to be greater than thehydrophobic threshold pressure. When the pressure is high at the sourcecavity end of a channel and low at the destination cavity end, fluidflows in the channel from the source cavity to the destination cavity.Intermediate gas pressure is insufficient to overcome the hydrophobicthreshold, but if fluid is already flowing (e.g., by subjecting thesource cavity to high gas pressure), intermediate gas pressure issufficient to continue allowing the fluid flow, albeit at a reducedrate. If fluid is already flowing and the pressure is reduced tointermediate gas pressure at the source cavity end of a channel andremains low at the destination end of the channel, fluid continuesflowing in the channel from the source cavity to the destination cavity,but air is not introduced into the channel.

The hydrophobic threshold pressure of hydrophobic channels keeps fluidin nodes and reservoirs from leaking into the channels when no pressuredifferences are applied. The threshold pressure is designed to be greatenough to prevent fluid flow that might be driven by the hydrodynamicpressure caused by the weight of fluid in a reservoir or node, or byresidual pressure differences that might exist when applied pressuresare switched between high and low. Thus a “hydrophobic channel” isdefined as one that exhibits a pressure threshold that prevents fluidfrom leaking into the channel when the pressure difference between thetwo ends of the channel is less than a designated or threshold pressure.In an example valveless microfluidic system, channels were designed tohave about 1 kPa hydrophobic threshold pressure.

One implementation of a valveless microfluidic flowchip includes asubstrate layer, a hydrophobic fluid layer, and a pneumatic layer. FIG.2 illustrates a valveless microfluidic flowchip, seen in cross section.In FIG. 2 , microfluidic flowchip 105 includes a substrate layer 110, ahydrophobic fluidic layer 115, and a pneumatic layer 120. Cavities inthe hydrophobic fluidic layer are labeled A, B and C in FIG. 2 and inFIGS. 3A-3F. Cavities A and B are connected by channel 125 whilecavities B and C are connected by channel 130. Cavities A and C areclassified as reservoirs because they are connected to only one channeleach. Cavity B is classified as a node because it is connected to morethan one channel: B is connected to both channel 125 and channel 130.

Pressure sources 135, 140 and 145 are connected to reservoir A, node Band reservoir C, respectively, via gas tubes 150, 155 and 160,respectively. Each of the three pressure sources is capable of providingat least two different pressures: a high gas pressure and a lowpressure. Labels HP, IP and LP in FIG. 2 and in FIGS. 3A-3F refer to thecapability of a pressure source to provide high, intermediate or lowpressures, respectively. Pressure source 145 is also capable ofproviding a pressure that is less than atmospheric pressure; e.g., apartial vacuum.

Several different ways of making a structure like microfluidic flowchip105 are possible. As a first example, substrate 110 may be made ofglass, polydimethylsiloxane (PDMS), polyethylene terephthalate (PET), orplastic. Hydrophobic fluidic layer 115 may be made from PDMS. A mold forcasting PDMS to define hydrophobic microfluidic channels may be producedwith a programmable cutter for vinyl decals or definedphotolithographically in an epoxy-based negative photoresist such asSU-8. After patterned PDMS is cured and removed from a mold, it may bebonded to a flat substrate. Pneumatic layer 120 may also be made fromPDMS. Gas tubes may be made from polyetheretherketone (PEEK) tubingwhich forms convenient seals when inserted in appropriately sized holesin PDMS. Hydrophobic materials that are suitable alternatives to PDMSinclude polypropylene (PP), a cyclic olefin polymer (COP), a cyclicolefin copolymer (COC), fluorinated ethylene propylene (FEP) andpolytetrafluoroethylene (PTFE). Published water contact angles for thesematerials are provided in Table 1.

TABLE 1 Critical Water Surface Contact Tension Angle Polymer Name(dynes/cm) (deg) Cyclic Olefin Polymer (COP)/ 30   88   Cyclic OlefinCopolymer (COC) Polypropylene 31.6  102.1  Polydimethylsiloxane 20.1 107.2  Fluorinated ethylene propylene 10.1  108.5 Polytetrafluoroethylene 10.4  109.2 

In some embodiments, the polymers can be modified to increase theirhydrophobicity through use of additives, surface coatings, or surfacemodifications.

In example microfluidic flowchips, the cross-sectional dimensions ofchannels 125 and 130 can be in the range of about 25 μm to about 50 μm,100 μm, 150 μm, or 200 μm (height) by about 25 μm to about 50 μm, 100μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, or 500 μm(width). The sizes of reservoirs A and C, and of node B can be betweenabout 1 mm to about 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5mm, or 6 mm in diameter. The distance between reservoir A and node B canbe between about 5 mm to about 25 mm, 50 mm, 75 mm or 100 mm; thedistance between node B and reservoir C can be within the same range.The cross-sectional areas of the cavities in typical flowchips areapproximately 100 to 400 times greater than the cross-sectional areas ofthe channels. Therefore the flow resistance of the channels is about10,000 to 160,000 times greater than the flow resistance of thecavities. Alternative designs for channels and cavities including fluidflow barrier structures or configurations lead to the flow resistance ofsuch channels being about 20%, 50%, 100%, 200%, 500%, or 1000% greaterthan the flow resistance of non-altered channels.

Another way to make a structure like microfluidic flowchip 105 involveshot embossing a hydrophobic thermoplastic polymer such as polypropylene(PP) or cyclic olefin polymer/copolymer (COP/COC) followed bysolvent-assisted lamination to form enclosed, hydrophobic channels. Athird way to make a structure like microfluidic flowchip 105 isinjection molding a hydrophobic polymer such as PP, COP or COC. Finally,hydrophilic microfluidic channels, formed in polycarbonate for example,may be made hydrophobic via chemical surface treatment. There are, nodoubt, other ways to make a structure containing cavities connected byhydrophobic microfluidic channels.

In some embodiments, one or more or a plurality of the cavities can beconnected with up to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16channels each. In some embodiments, each network in the one or morenetworks comprises an input/output channel, the input/output channelhaving a greater resistance to fluid flow than that of the microfluidicchannels.

3. Systems Comprising Valveless Microfluidic Flowchips

Further provided are systems comprising valveless microfluidicflowchips, including those known in the art and the valvelessmicrofluidic flowchips described above and herein. Additionally, thesystems comprise a pressure sequencer connected by pneumatic deliverychannels to: (1) a high gas pressure gas source; (2) an intermediate gaspressure gas source; (3) a low pressure gas source; and optionally, (4)a partial vacuum pressure gas source; and to at least one cavity, e.g.,at least two cavities, in the flowchip.

In some embodiments, the pressure sequencer is configured to apply ahigh gas pressure, an intermediate gas pressure, a low gas pressure, andoptionally, a partial vacuum pressure to at least one cavity accordingto pressure sequence data, where the high gas pressure is greater thanthe intermediate gas pressure, the intermediate gas pressure is greaterthan the low gas pressure, and the low gas pressure is greater than thepartial vacuum gas pressure, and the partial vacuum pressure is lessthan atmospheric pressure. In implementing the present systems, theflowchip can but need not additionally comprise microfluidic channelscomprising a hydrostatic resistance barrier. In some embodiments, thepressure sequencer is configured to concurrently apply a combination ofgas pressure and partial vacuum to at least one cavity.

Fluid transfer between cavities, e.g., between reservoirs and nodes isaccomplished by switching pressures applied to each reservoir and nodein a system according to a specific pattern. The following terminologyaids discussion of a fluid transfer rule for the present valvelessmicrofluidic systems. The origin or source cavity is a reservoir or nodefrom which fluid is to be transferred. The destination cavity is thereservoir or node to which fluid is to be transferred. In someembodiments of the present systems, at least three gas pressures areused: high gas pressure, intermediate gas pressure and low gas pressure.In some embodiments of the present systems, at least four gas pressuresare used: high gas pressure, intermediate gas pressure, low gas pressureand partial vacuum.

A fluid transfer rule for the present valveless microfluidic systems maybe summarized in the following steps:

Step 0: Apply low pressure to all cavities.

Step 1: Apply high gas pressure to the origin or source cavity and anycavity connected to the origin or source cavity by a microfluidicchannel, other than the destination cavity for a time t(1) which is atime period that is stopped or ended before the quantity of liquid iscompletely removed from the source cavity, e.g., a time periodsufficient to allow at least about 10% and up to about 20%, 30%, 40%,50%, 60%, 70%, 80% or 90% of the total volume of fluid in the origin orsource cavity to drain into the microfluidic channel connecting thesource cavity with the destination cavity. Apply low pressure to thedestination and any cavity connected to the destination, other than theorigin.

Step 2: Apply intermediate gas pressure to the origin or source cavityfor a time t(2) sufficient to push or expel all the remaining fluid inthe source cavity to drain into the microfluidic channel connecting thesource cavity with the destination cavity. The application ofintermediate gas pressure on the source cavity is insufficient pressureto force air into the connecting microfluidic channel. No air gap isintroduced into the microfluidic channel.

Step 3: (Optional) Apply partial vacuum to the destination cavity for atime t(3) sufficient to evacuate all fluid from the cavity. This can bedone with or without applying pressure to other wells depending on thedesired extent of fluid removal.

Step 4: Return to Step 0 to prepare for the next fluid transferoperation.

As explained herein, the fluid transfer rule may be executed by apressure sequencer that is configured to execute the required sequenceof pressures to accomplish any desired fluid transfer operation. Thepressure sequencer receives pressure sequence data and/or instructionsfrom, e.g., a controller. These data or instructions includes step bystep instructions specifying what pressure is to be applied to eachreservoir and node in device in order to carry out a specific fluidtransfer operation. Fluid can be moved from any reservoir to any otherreservoir in a reconfigurable microfluidic system by repeating the stepsof the fluid transfer rule.

In some implementations, a controller is part of a microfluidics systemas described herein. Such system may be integrated with electronics orother processing logic for controlling their operation before, during,and after processing by a pressure sequencer. The processing logic maybe referred to as the “controller,” which may control various componentsor subparts of the system or systems. The controller, depending on theprocessing requirements and/or the type of system, may be programmed tocontrol any of the processes disclosed herein, including the delivery ofgases, pressure settings, vacuum settings, power settings, flow ratesettings, fluid delivery settings, volume settings, positional andoperation settings connected to or interfaced with a specificmicrofluidics system.

The controller may be implemented in any of various integrated circuits,logic, memory, and/or software that receive instructions, issueinstructions, control operation, enable endpoint measurements, and thelike. The integrated circuits may include chips in the form of memorythat store program instructions, digital signal processors (DSPs), chipsdefined as application specific integrated circuits (ASICs), and/or oneor more microprocessors, or microcontrollers that execute programinstructions (e.g., software). Program instructions may be instructionscommunicated to the controller in the form of various individualsettings (or program files), defining operational parameters forcarrying out a particular process. The controller may have access tocomputer readable media such as storage media, computer storage media,or data storage devices (removable and non-removable) such as, forexample, magnetic discs, optical disks, or tape. Computer storage mediamay include volatile and non-volatile, removable and non-removable mediaimplemented in any method or technology for storage of information, suchas computer readable instructions, data structures, program modules, orother data. Examples of computer storage media include RAM, ROM, EEPROM,flash memory or other memory technology, CD-ROM, digital versatile discs(DVD) or other optical storage, magnetic cassettes, magnetic tape,magnetic disk storage or other magnetic storage devices, or any othermedium which can be used to store the information and which can beaccessed by an application, module, or both. Any such computer storagemedia may be part of the device or accessible or connectable thereto.The computer storage media may be located remotely, e.g., cloud storage,and accessed via a network or internet connection. Any method,application or module herein described may be implemented using computerreadable/executable instructions that may be stored or otherwise held bysuch computer readable media and executed by the one or more processors.

The controller, in some implementations, may be a part of or coupled toa computer that is integrated with, coupled to the system, otherwisenetworked to the system, or a combination thereof. For example, thecontroller may be in the “cloud” or all or a part of a host computersystem, which can allow for remote access of the pressure sequencer. Insome embodiments, the host computer system and/or the controller can beconnected to the internet (e.g., via a wired or wireless connection).The computer may enable remote access to the system to monitor currentprogress of fluidic operations, examine a history of past pressuresequencing operations, examine trends or performance metrics from aplurality of pressure sequencing operations, to change parameters ofcurrent processing, to set processing steps to follow a currentprocessing, or to start a new process. In some examples, a remotecomputer (e.g. a server) can provide pressure sequencing recipes to asystem over a network, which may include a local network or theinternet. The remote computer may include a user interface that enablesentry or programming of parameters and/or settings, which are thencommunicated to the system from the remote computer. In some examples,the controller receives instructions in the form of data, which specifyparameters for each of the processing steps to be performed during oneor more operations. It should be understood that the parameters may bespecific to the type of process to be performed and the type of toolthat the controller is configured to interface with or control. Thus asdescribed above, the controller may be distributed, such as bycomprising one or more discrete controllers that are networked togetherand working towards a common purpose, such as the processes and controlsdescribed herein. An example of a distributed controller for suchpurposes would be one or more integrated circuits locally associatedwith one or more pressure sequencers in communication with one or moreintegrated circuits located remotely (such as part of a remote computer)that combine to control one or more pressure sequences.

As noted above, depending on the process step or steps to be performedby the pressure sequencer, the controller might communicate with one ormore of other pressure sequencers in fluid communication with one ormore microfluidic chips, in sequence or in parallel, a main computer, oranother controller.

In addition to pressure sequencing, the controller may assist indetection of assay parameters (e.g., reservoir pressures, reservoirvolumes, fluid flow rate), and biomarker detection (e.g., whenperforming an immunoassay). In some cases, the controller may host auser-accessible platform for invoking services, such as reporting andanalysis services, and for providing computational resources to effectmachine learning techniques on the detection data.

In various embodiments, the pressure sequencer can be implemented as aset of electronically controlled pneumatic valves, e.g., that areprogrammed using software (e.g., LabVIEW, National InstrumentsCorporation, MATLAB, Mathworks, Visual BASIC, C#, Python, or Java)),e.g., running on a personal computer, a microcontroller ormicroprocessor. In various embodiments, the pressure sequence datanecessary to move fluid from one reservoir to another in areconfigurable microfluidic device can be programmed or worked outmanually. In various embodiments, a graphical software program may bewritten that allows a user to select origin and destination reservoirs,with the program then generating appropriate pressure sequence data byrepeated application of the fluid transfer rule. In this way anintuitive system may be created that permits users to perform arbitrarymicrofluidic experiments without needing to understand the fluidtransfer rule or other system operation details.

Examples herein show how the fluid transfer rule is used to performcommon fluid transfer experiments.

FIGS. 3A-3F illustrate an implementation using the herein describedsystems and flowchips for transferring fluid from a source cavity (A) toa destination cavity (B) through a connecting channel. A high gaspressure (HP) is applied for a time t(1) to overcome the hydrostatic andhydrophobic barriers between the source cavity (A) and connected channeland start fluid flowing through the channel to the destination cavity(B) (See, e.g., FIGS. 3A-3C). The pressure on the source cavity (A) isthen switched to an intermediate gas pressure (IP) for a time t(2) thatwill continue to move fluid through the channel and empty the sourcecavity (A) (See, e.g., FIGS. 3D-3E). The force exerted by IP is lessthan the amount required to overcome the resistance or fluid flowbarrier(s) at the channel/cavity interface when the source cavity (A)has emptied so fluid is not pushed down the channel. The destinationcavity (B) is kept at low pressure (LP, atmospheric or ambient) duringthis transfer. At the end of this transfer event, the source cavity (A)is empty, the connecting channel is full, and the destination cavity (B)has been filled with fluid (See, e.g., FIG. 3F). The total volume in thedestination cavity (B) is the volume in the source cavity (A) minus thevolume in the channel. The time t(1) is set so that at least about 10%up to about 70%, 75%, 80%, 85% or 90%, e.g., between about 30% and about70%, of the fluid in the source cavity (A) has been transferred. Thetime t(2) is set for a time period that is longer than the time requiredto transfer the remaining fluid in the source cavity (A).

FIGS. 4A-4F illustrate an implementation using the herein describedsystems and flowchips for transferring fluid from a source cavity (B) toa destination cavity (C) through a connecting channel and thenevacuating or removing fluid from the destination cavity (C). A highpressure (HP) is applied for a time t(1) to cavities A and B to overcomethe fluid flow barriers between the source cavity (B) and channelconnected to cavity C and start fluid flowing through the channel to thedestination cavity (C) (See, e.g., FIGS. 4A-4B). In this scenario fluidwill remain in cavity A. The pressure on cavities A and B is thenswitched to an intermediate pressure (IP) for a time t(2) that willcontinue to move fluid through the B-C channel and empty the sourcecavity (B) (See, e.g., FIG. 4C). The force exerted by IP is less thanthe amount required to overcome the resistance or fluid flow barrier(s)at the channel/cavity interface when the source cavity (B) has emptiedso fluid is not pushed down the channel. The destination cavity (C) iskept at low pressure (LP, atmospheric or ambient) during this transfer.At the end of this transfer event, cavity A remains full, the sourcecavity (B) is empty, the connecting channel is full, and the destinationcavity (C) has been filled with fluid (See, e.g., FIG. 4C). The totalvolume in the destination cavity (C) is the volume in the source cavity(B) minus the volume in the channel. The time t(1) is set so that atleast about 10% up to about 70%, 75%, 80%, 85% or 90%, e.g., betweenabout 30% and about 70%, of the fluid in the source cavity (B) has beentransferred. The time t(2) is set for a time period that is longer thanthe time required to transfer the remaining fluid in the source cavity(C). The pressure on the source cavity (C) is then switched to partialvacuum (VAC) for a time t(3) and fluid is removed from the source cavity(C) and channel B-C through the gas port (See, e.g., FIGS. 4D-4E). Atthe end of this event, cavity A remains full and the source cavity (B),channel B-C, and destination cavity (C) are empty (See, e.g., FIG. 4F).

Use of a single gas port to both apply pressure to and evacuate fluidfrom a cavity has certain limitations. If residual fluid remains in agas line after an evacuation step, then that fluid can be pushed downinto the cavity during a subsequent step when pressure is applied andgas moves through that gas line into the cavity. This can introduceundesirable effects such as cross-contamination. Furthermore, the designof a manifold and sealing to a flowchip is more complex when it needs tohandle both pressure and partial vacuum. Such a system is more prone topressure leakage over multiple fluid transfer cycles which can lead tofluid transfer errors. An improved system, shown in FIG. 5 , hasseparate gas ports for applying pressure to and evacuating fluid from acavity. A second manifold is interfaced to the bottom of the flowchip. Agas port is connected to this manifold at one or more cavities.Connection of the cavity to the manifold can be done through pre-formedholes in the bottom seal of the cavity or by penetration through thebottom seal of the flowchip when the flowchip is mounted on the bottommanifold (e.g., with a hollow needle). The bottom manifold can beconnected to the same pressure and partial vacuum sources used for thetop manifold or have separate pressure and partial vacuum sources.

FIGS. 6A-6F illustrate an implementation using the herein describedsystems and flowchips for transferring fluid from a source cavity (B) toa destination cavity (C) through a connecting channel and thenevacuating or removing fluid from the destination cavity (C) wheredestination cavity (C) has separate gas ports for applying pressure andevacuating fluid. A high pressure (HP) is applied for a time t(1) tocavities A and B to overcome the hydrostatic and hydrophobic barriersbetween the source cavity (B) and channel connected to cavity C andstart fluid flowing through the channel to the destination cavity (C)(See, e.g., FIGS. 6A-6B). In this scenario fluid will remain in cavityA. The pressure on cavities A and B is then switched to an intermediatepressure (IP) for a time t(2) that will continue to move fluid throughthe B-C channel and empty the source cavity (B) (See, e.g., FIG. 6C).The force exerted by IP is less than the amount required to overcome theresistance or fluid flow barrier(s) at the channel/cavity interface whenthe source cavity (B) has emptied so fluid is not pushed down thechannel. The destination cavity (C) is kept at low pressure (LP,atmospheric or ambient) during this transfer. At the end of thistransfer event, cavity A remains full, the source cavity (B) is empty,the connecting channel is full, and the destination cavity (C) has beenfilled with fluid (See, e.g., FIG. 6C). The pressure on the bottom gasport of source cavity (C) is then switched to partial vacuum (VAC) whilethe pressure on the top gas port is kept at low pressure for a time t(3)and fluid is removed from the source cavity (C) through the bottom gasport (See, e.g., FIGS. 6D-6E). At the end of this event, cavity Aremains full, the channel B-C remains full, and the source cavity (B)and destination cavity (C) are empty (See, e.g., FIG. 6F).

Accordingly, provided is a system for moving a quantity of liquid from asource cavity to a destination cavity and evacuation of fluid from thedestination cavity in a network of microfluidic cavities, wherein thesource cavity and the destination cavity are separated by a valvelessmicrofluidic channel having a resistance to fluid flow greater than thatof the source cavity, the method comprising: (i) a receptacle forreceiving and engaging with a flowchip comprising the network ofmicrofluidic cavities; (ii) a pressure sequencer comprising a set of gasvalves and configured to be connected to a first gas source forproducing a high pressure in microfluidic cavities, a second gas sourcefor producing a low pressure in microfluidic cavities, and a third gassource for producing an intermediate pressure in microfluidic cavities,and optionally a fourth source for producing a partial vacuum, whereinthe high pressure is greater than the low pressure, the intermediatepressure is less than the high pressure but greater than the lowpressure, and the intermediate pressure is insufficiently great toovercome resistance or barrier(s) to fluid flow in the microfluidicchannel when the source cavity is substantially empty of the liquid,wherein the pressure sequencer can apply any pressure state to anycavity; and (iii) a controller configured to direct the pressuresequencer to: (a) apply the high pressure to the source cavity and toall other cavities connected to the source cavity excepting thedestination cavity, while applying the low pressure to the destinationcavity, to move a portion of the quantity of liquid from the sourcecavity, through the microfluidic channel, and to the destination cavity,and (b) apply an intermediate pressure to the source cavity before thequantity of liquid is completely removed from the source cavity, whereinthe intermediate pressure is sufficiently great to push at least some ofthe quantity of liquid remaining after (a) to the destination cavity,but avoids introducing gas into the microfluidic channel, and (c)optionally apply a partial vacuum to evacuate fluid from one or morecavities. While not wishing to be bound by any theory, it is believedthat an air-liquid interface at the entrance to the microfluidic channel(adjacent the source cavity) provides an increased resistance orbarrier(s) to fluid flow that prevents further fluid transfer when thesource cavity is first emptied (or substantially emptied) of the liquid.Thus, the intermediate pressure is sufficient to push fluid out of thesource cavity only so long as there is fluid in the cavity. When thatcavity is emptied, the resistance to fluid transfer increases such theintermediate pressure is no longer sufficient to drive fluid through thechannel.

In some embodiments, the pressure sequencer is configured to apply orfollow a fluid transfer rule in which: (1) high gas pressure is appliedto an origin or source cavity from which a fluid is transferred and lowgas pressure is applied to a destination cavity to which the fluid istransferred, the high pressure being applied for a time t(1) sufficientto overcome hydrophobic and/or hydrostatic barriers and start fluidflowing from the origin or source cavity into a microfluidic channelconnecting the origin or source cavity to the destination cavity; (2)intermediate pressure is applied to the origin or source cavity and lowpressure is applied to the destination cavity such that fluid continuesto move through the connecting channel, the intermediate pressure beingapplied for a time sufficient to empty the origin or source cavity offluid but of a pressure insufficient to expel fluid out of the channel;whereby the origin or source cavity is emptied of fluid and the fluid ismoved into the channel and destination cavity; and (3) optionally,partial vacuum is applied to the destination channel while low pressureis applied to the source cavity such that fluid is evacuated from thedestination cavity through the gas port. In some embodiments, partialvacuum is applied to the destination cavity through a separate port orchannel located on the bottom surface of the destination cavity 220, oropposite side of the pressure ports, e.g., so that less stress isapplied to the manifold/flowchip interface, and fluid is evacuated fromthe bottom of the cavity. In some embodiments, gas pressure isintroduced into the destination cavity from the gas port above the topsurface of the flowchip to facilitate removal of fluid from, and dryingof the destination cavity by the partial vacuum port below the flowchip.As used herein, the terms “above” and “below” are relative because theflowchip could be held in a vertical configuration. Gas pressure isapplied above the meniscus of the fluid in the destination cavity andpartial vacuum is concurrently applied below the fluid in thedestination cavity, e.g., on opposite sides of the fluid in thedestination cavity, facilitating evacuation with continuous flow offluid.

While not wishing to be bound by any theory, it is believed that anair-liquid interface at the entrance to the microfluidic channel(adjacent the source cavity) provides an increased resistance orbarrier(s) to fluid flow that prevents further fluid transfer when thesource cavity is first emptied (or substantially emptied) of the liquid.Thus, the intermediate gas pressure is sufficient to push fluid out ofthe source cavity only so long as there is fluid in the cavity. Whenthat cavity is emptied, the resistance to fluid transfer increases suchthe intermediate gas pressure is no longer sufficient to drive fluidthrough the channel.

In some embodiments of the systems, the pressure sequencer is configuredto apply one or more pressure modes selected from the group consistingof constant pressure, pulsing pressures, increased ramping pressures anddecreased ramping pressures. In some embodiments, the pressure sequenceris configured to control applied pressure by applying pulsing pressuresand using pulse width modulation (PWM), which may have a duty factorchosen to provide the desired pressure. For example, during operation,the duty factor may be adjusted to being in the range of about 1%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In some embodiments, thepressure sequencer is configured to apply increased and/or decreasedramping pressures comprising rise and/or fall times in the range ofabout 10 msec to about 20 msec, 50 msec, 100 msec, 250 msec, 500 msec,750 msec or 1 sec.

As an example, high pressure can be in the range of about 5 kPa to about100 kPa, intermediate pressure can be in the range of about 1.0 kPa toabout 10 kPa, low pressure can be about 0 kPa or atmospheric or ambientpressure, and partial vacuum pressure can be less than atmosphericpressure, e.g., about −6 kPa or lower, where all pressures are gaugepressures. In some embodiments, the high pressure is in the range ofabout 5 kPa to about 60 kPa, 70 kPa, 80 kPa, 90 kPa or 100 kPa, e.g., inthe range of about 10 kPa to about 60 kPa. In some embodiments, theintermediate pressure is in the range of about 1 kPa to about 5 kPa, 6kPa, 7 kPa, 8 kPa, 9 kPa or 10 kPa. In some embodiments, the partialvacuum pressure is in the range of about −5 kPa to about −10 kPa, −20kPa, −30 kPa, −40 kPa, −50 kPa, −60 kPa, −70 kPa, −80 kPa, −90 kPa, or−100 kPa. In some embodiments, fluid flow rate under high gas pressurethrough the first plurality of microfluidic channels is from about 0.1μL/second to about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0,3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10.0 μL/second. In someembodiments, fluid flow rate under intermediate pressure through thefirst plurality of microfluidic channels is from about 0.01 μL/second toabout 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0μL/second. In some embodiments, a plurality of the microfluidic channelspresent a hydrophobic pressure barrier to fluid flow that is less thanthe pressure difference between the high gas pressure and the low gaspressure.

In some embodiments, the pressure sequencer is configured to apply orfollow a fluid transfer rule in which: (1) high gas pressure is appliedto an origin or source cavity from which a fluid is transferred and lowgas pressure is applied to a destination cavity to which the fluid istransferred, the high gas pressure being applied for a time t(1)sufficient to overcome hydrophobic and/or hydrostatic barriers and startfluid flowing from the origin or source cavity into a microfluidicchannel connecting the origin or source cavity to the destinationcavity; and (2) intermediate gas pressure is applied to the origin orsource cavity and low pressure is applied to the destination cavity suchthat fluid continues to move through the connecting channel, theintermediate gas pressure being applied for a time t(2) sufficient toempty the origin or source cavity of fluid but of a pressureinsufficient to expel fluid out of the channel; whereby the origin orsource cavity is emptied of fluid and the fluid is moved into thechannel and destination cavity. In some embodiments, time t(1) is for atime period that is stopped or ended before the quantity of liquid iscompletely removed from the source cavity, e.g., a time periodsufficient to drain at least about 10% and up to about 90% of the fluidvolume from the origin or source cavity. In some embodiments, partialvacuum is applied to the destination cavity through a separate port orchannel located on the bottom surface of the destination cavity 220, oropposite side of the pressure ports, e.g., so that less stress isapplied to the manifold/flowchip interface, and fluid is evacuated fromthe bottom of the cavity. In some embodiments, gas pressure isintroduced into the destination cavity from the gas port above the topsurface of the flowchip to facilitate removal of fluid from, and dryingof the destination cavity by the partial vacuum port below the flowchip.Again, the terms “above” and “below” are relative because the flowchipcould be held in a vertical configuration. Gas pressure is applied abovethe meniscus of the fluid in the destination cavity and partial vacuumis concurrently applied below the fluid in the destination cavity, e.g.,on opposite sides of the fluid in the destination cavity, facilitatingevacuation with continuous flow of fluid.

In some embodiments, the pressure sequencer is further connected to avery high gas pressure source, and the pressure sequencer is configuredto apply a very high gas pressure, wherein the very high gas pressure isgreater than the high gas pressure. In some embodiments, the very highgas pressure is at least about 100 kPa, e.g., at least about 125 kPa,150 kPa, 175 kPa, 200 kPa, or higher.

In some embodiments, the pressure sequencer is configured to apply orfollow a fluid transfer rule in which the partial vacuum gas pressure isapplied to a destination cavity to which a fluid is drawn via itsinput/output channel and low gas pressure is applied to any other cavityconnected to the destination cavity by a channel.

In some embodiments, one or more networks comprise j rows and k columnsof cavities, j and k being positive integers, cavities in each row orcolumn being connected in series.

4. Methods of Use

In a further aspect, provided are methods of moving a quantity of liquidfrom a source cavity to a destination cavity in a network ofmicrofluidic cavities. The methods are applicable for use in thevalveless microfluidic flowchips and applying the microfluidic systemsdescribed herein, and in currently available valveless microfluidicflowchips and systems. In some embodiments, the methods employ amicrofluidic flowchip having a source cavity and a destination cavityseparated by a valveless microfluidic channel having a resistance tofluid flow greater than that of the source cavity. In some embodiments,the methods comprise: (a) applying a high gas pressure to the sourcecavity, and all other cavities connected to the source cavity exceptingthe destination cavity, while applying a low pressure to the destinationcavity to move a portion of the quantity of liquid from the sourcecavity, through the microfluidic channel, and to the destination cavity,wherein the high gas pressure is greater than the low pressure; and (b)applying an intermediate gas pressure to the source cavity before thequantity of liquid is completely removed from the source cavity, whereinthe intermediate gas pressure is lower than the high gas pressure buthigher than low pressure, and wherein the intermediate gas pressure issufficiently great to push at least some of the quantity of liquidremaining after (a) to the destination cavity, but insufficiently greatovercome resistance to fluid flow in the microfluidic channel, andthereby avoid introducing gas into the microfluidic channel. In someembodiments, the one or more of the microfluidic channels arehydrophobic or comprise a hydrophobic coating. In some embodiments, theintermediate gas pressure is insufficiently great to introduce gas intothe microfluidic channel even when all of the quantity of liquid hasbeen removed from the source cavity. In some embodiments, less thanabout 90% of the liquid is removed from the source cavity beforeapplying the intermediate gas pressure. In some embodiments, a definedamount of fluid remains in the source cavity in a region between theentrance and exit channels. In some embodiments, the method is performedusing a system as described above and herein.

In a further aspect, provided are methods for arranging fluid in amicrowell plate. In some embodiments, the methods comprise operating thevalveless microfluidic system as described above and herein according toa set of pressure sequence data that causes the fluid to be drawn intothe system from an origin or source cavity of the microwell plate andexpelled into a destination cavity of the microwell plate, wherein airis not introduced into a microfluidic channel downstream of an origin orsource cavity.

In a further aspect, provided are methods for performing a homogenousassay with j samples and k reagents. In some embodiments, the methodscomprise operating the valveless microfluidic system as described aboveand herein, with pressure sequence data that causes each of the jsamples to be exposed to the k reagents thereby producing j outputsolutions, wherein air is not introduced into a microfluidic channeldownstream of an origin or source cavity.

In a further aspect, provided are methods for performing a multiplexedimmunoassay. In some embodiments, the methods comprise operating thevalveless microfluidic system as described above and herein, wherein thesystem comprises two or more networks, the system operated according topressure sequence data such that the pressure sequencer directs fluidflows in the system that cause different kinds ofsample-analyte-capture-analyte reactions to occur in different networks,but the same kind of detection reagent reaction to occur in a pluralityof networks, wherein air is not introduced into a microfluidic channeldownstream of an origin or source cavity. In some embodiments, theimmunoassay fluid comprises a buffer having a pH in the range of 6-11,e.g., pH in the range of 6-9, e.g., a pH in the range of about 7-9 or apH in the range of 9-11, one or more blocking agents or proteinsolutions and one or more surfactants. In specific embodiments, theimmunoassay fluid comprises phosphate buffered saline (PBS),tris-buffered saline (TBS) or a bicarbonate buffer, albumin (e.g.,bovine serum albumin (BSA)), Tween-20, Triton-X, or other surfactantsand optionally glycerol.

In some embodiments, the methods can be executed analogously to themethods described in U.S. Patent Publication Nos. US2017/0021351,US2017/0021352 and US2017/0021353, with the improvement that thepressure sequencer is configured to switch from high gas pressure modeto intermediate gas pressure mode before the quantity of liquid iscompletely removed from the source cavity, thereby avoiding introductionof air bubbles into the microfluidic channel that connects the origincavity with the destination cavity.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Methods and Valveless Microfluidic Flowchips for ImprovedFluid Control

This example illustrates implementation for transferring fluid from asource cavity to a destination cavity through a connecting channel. Aschematic representation of this process is shown in FIGS. 3A-3F. A highgas pressure (HP), e.g., in the range of about 5 kPa or 10 kPa to about60 kPa or 100 kPa, is applied for a time t(1) to overcome thehydrophobic and hydrostatic barriers between the source cavity andconnected channel and start fluid flowing through the channel to thedestination cavity (FIGS. 3A-3C). The pressure on the source cavity isthen switched to a second, intermediate gas pressure (IP), e.g., in therange of about 0.5 kPa or 1.0 kPa to about 5 kPa or 10 kPa, for a timet(2) that will continue to move fluid through the channel and empty thesource cavity (FIGS. 3D-3E). The force exerted by an intermediate gaspressure (IP) is less than the amount required to overcome theresistance or fluid flow barrier(s) at the cavity/channel interface whenthe source cavity has emptied so fluid is not pushed down the channel.The destination cavity is kept at a low pressure (LP, e.g., atmospheric)during this transfer. At the end of this transfer event, the sourcecavity is empty, the connecting channel is full, and the destinationcavity has been filled with fluid (FIG. 3F). The total volume in thedestination cavity is the volume in the source cavity minus the volumein the channel. The time t(1) is set so that 10% to 90% of the fluid inthe source cavity has been transferred. The time t(2) is set so that ismuch longer than the time required to transfer the remaining fluid inthe source cavity.

In one implementation using a polypropylene (PP) flowchip with channeldimensions 200 μm×50 μm×25 mm (W×H×L) the HP=30 kPa and IP=1.5 kPa whichgives flow rates through the channel of about 2 μl/sec and about 0.1μl/sec respectively. For a transfer of 20 μl, t(1)=7 sec and t(2)=120sec. Nominally, 14 μl of fluid is transferred by HP and 6 μl of fluid byIP. The fluid should be completely transferred during the IP step afterabout 60 sec. The excess IP time accommodates for variation in fluidtransfer rates caused by channel dimensional variations, presence ofartifacts or contamination in channels, presence of air bubbles inchannels, or other effects. The times t(1) and t(2) are configured sothat the source cavity will not empty during the HP step and the totaltransfer time is minimized. The measured flowrate variation overmultiple channels and multiple flowchips is approximately 12%, whichgives a “3-sigma” maximum HP flowrate of 2.72 μl/sec. Under the aboveconditions the maximum amount of fluid transferred during HP will be 19μl so the source cavity will not be emptied. The “3-sigma” minimum HPflowrate is 1.28 μl/sec, making the expected minimum amount of fluidtransferred to be 9 μl. This means 11 μl will be transferred at the IPrate which will take 110 sec which is less than t(2). This methodassures that all of the fluid will be transferred out of the sourcecavity, but air will not be forced through the channel and into thedestination cavity. The time t(2) can be increased if desired toaccommodate variations in the IP flowrate.

The resistance at the WCI is a function of the surface and fluidproperties and the channel dimensions. The rectangular cross-sectionfluidic resistance formula is:

$R_{h} = {\frac{12\mu L}{\omega{h^{3}\left( {1 - \frac{0.63h}{\omega}} \right)}}.}$

In this formula μ: fluid viscosity; L: channel length; ω: channel width;and H: channel height. The fluid viscosity can be optimized to increasethis resistance and allow higher values of IP to be used in the process.Additives such as glycerol, and other higher viscosity fluids have beenmixed into assay reagents in order to increase this resistance. Thesefluids will evacuate from channels at higher IP values. A useful “assaybuffer” solution for flowchips made from polypropylene (PP) containsPBS+0.1% BSA+0.001% Tween 20. A useful “assay buffer” solution forflowchips made from cyclic olefin copolymer (COC) contains PBS+0.1%BSA+0.001% Tween 20+10% glycerol.

In addition, the channel geometry can be modified to increase thehydrostatic barrier (HSB) for example by introducing a “neck” or a“serpentine” structure at or near the WCI. Examples of these are shownin FIGS. 7A-7C. The HSB pressures were measured for polydimethylsiloxane(PDMS) devices with these geometries and the results are given in Table2, below, and which is also depicted in FIG. 8 .

TABLE 2 HPB (kPa) HSB (kPa) Straight 1.3 2.0 Neck 2.1 4.0 Serpentine 3.03.8 HPB-Hydrophobic Barrier HSB-Hydrostatic Barrier

The hydrostatic resistance or fluid flow barrier structures are designedso that there is an increase in both the hydrophobic barrier (HPB),which relates to the resistance of liquid moving from a cavity into achannel, and the hydrostatic barrier (HSB), which relates to theresistance of moving liquid from a channel. It is also critical tomaintain adequate flowrates so that the fluid transfers can be performedin a reasonable amount of time, however. This is especially importantfor time-sensitive steps in an immunoassay like the substrate incubationtime. The flowrates for an assay buffer were measured for the threestructures at different applied pressures and the results are shown inFIG. 8 . Some reduction in flowrates were observed, but were within anacceptable range.

In addition, the channel geometry can be modified to include a sealedcavity, or void, along the length of the channel between two regularcavities. An example of this is shown in FIGS. 9A-9B. A void ischaracterized by its diameter and height and the presence of a void in achannel leads to an increase in the breakthrough pressure (BP) of thechannel. The BP is defined as the pressure required to move fluid from asource cavity to a destination cavity. The BPs were measured forpolydimethylsiloxane (PDMS) devices with void diameters of 250 μm and500 μm, and void heights of 90 μm and 250 μm and the results are givenin FIG. 10 . In these cases the channel width and height were 50 μm.

In addition, the channel geometry can be modified to include a region ofreduced height (referred to herein as a rib or a rib feature), along thelength of the channel between two cavities. An example of this is shownin FIGS. 11A-11B. A rib is characterized by its length, width, andheight and the presence of a rib in a channel leads to an increase inthe breakthrough pressure (BP) and capillary pressure of the channel.The BPs were measured for polydimethylsiloxane (PDMS) devices withchannel widths of 50 μm and channel heights from 22 μm to 50 μm and theresults are given in FIG. 12 . The BPs were also compared to calculatedcapillary pressures for various rib geometries and those results aregiven in FIG. 13 . A good correlation (R2=0.927) was observed betweenthe BP and capillary pressure.

The mold for injection molded microfluidic devices is typically formedby sandwiching two sides together: an A-side and a B-side. A standardmethod is to have the A-side contain fluidic channel features and theB-side contain cavity and support features. Cavities are commonly formedby cylindrical pins and junctions between cavities and channels are madewhere the end of these pins press against raised features, or landingpads, on the A-side that define the bottom of the cavities and channelconnections. Limitations of alignment of the A-side and B-side requiresthat the landing pads be larger than the ends of the pins so that thereis always a complete connection (i.e., full contact between surfaces)between those items. An example of this is shown in FIG. 14A. The Leftimage in FIG. 14A shows a bottom view of the junction between a pin anda landing pad that has a single channel connection; the Middle imageshows a cross sectional view of that region; the Right image shows a3-dimensional top view of that region. A consequence of this assemblymethod is the formation of a lip which creates a microfluidic “ring” atthe base of a cavity that is nominally the same height as the connectingchannel (e.g., 50 μm). Fluid can be drawn through this ring bymicrocapillary forces and if two or more channels are connected to thesame cavity (e.g., a node) the ring can form a microfluidic connectionbetween those channels that can circumvent the hydrophobic barrierestablished between the cavity and channel. In addition, the connectiongeometry is flared with a radius of curvature defined by the machinetooling used to create the mold (see FIG. 14A—Right). This smoothing ofthe channel junction can reduce the fluid flow barrier at the WCI.

An improved device is shown in FIG. 14B. In this case, the B-side pin islarger than the A-side landing pad. This provides the necessarytolerance for alignment of the two mold sides while eliminating the lipat the base of the cavity formed by those items. This removesmicrofluidic connections between two or more channels that havejunctions with the same cavity. An additional improvement is thatchannels now go straight into the cavities making a sharp change ingeometry between the channel and cavity, because the junction of thechannel with the cavity is perpendicular. This increases fluid flowbarriers into and out of a cavity and improves the ability to controlfluid transfers.

The design in FIG. 14B has all the WCIs in one plane at the bottom of acavity. The lack of a microfluidic landing pad gap connection betweenchannels reduces potential wicking, but fluidic connections can still beformed between two or more channels that lead to adverse effects onassay performance (e.g., cross-contamination). A further improvement ofsuch junctions is shown in FIGS. 15A-15B. FIG. 15A shows a top3-dimensional view of a cavity with entrance ports close to the bottomof the cavity. FIG. 15B shows a bottom 3-dimensional view of channelsand their junctions with a cavity. FIG. 15C shows a cross sectional viewof a Transfer Channel entrance port and an exit junction to an AssayChannel. A feature of this device is the Transfer channels enter thecavity in a plane that is above the bottom of the cavity. Fluid thenexits the cavity into the Assay Channel at the bottom of the cavity.This provides a vertical separation between the junctions and furtherreduces the likelihood of fluidic connection between Transfer Channelsand the Assay Channel. The geometry of the Entrance Port is also suchthat more sharp edges are formed then in the case of the device in FIG.14B. This will further increase fluid flow barriers into and out ofthese channels and improve ability of the device to control fluidtransfers.

Improvement in fluid control provided by the cavity features shown inFIG. 15 was measured by observing passive leakage of a high surfactantfluid from a cavity into connecting channels. Images from this study areshown in FIGS. 16A and B. An aqueous solution with 0.1% Tween 20 andfluorescein dye (for visualization) was loaded into various cavities offlowchips and let to stand for 60 minutes. The bottoms of the cavitieswere imaged using a fluorescence microscope (Lumascope with 4×objective, 490 excitation, 530 nm emission from Etaluma, Carlsbad CA). Apositive result for passive leakage was determined if fluid was observedto travel more than 1 mm into the channel. The percentage of channelsexhibiting passive leakage was used to gauge the flowchip performance.For the device shown in FIG. 14B with native COC surfaces approximately50% of channels were observed to have passive leakage. The addition ofhydrophobic surface coatings reduced this to less than 17%. The deviceshown in FIG. 15 with native COC surfaces exhibited no passive leakage.

The method described in this example can be extended to use of ndifferent HP settings where n>2. This can allow for more exquisitecontrol of fluid movements for running assays in flowchips with a widevariety of cavity and channel dimensions and multiple fluid types. Forexample, multiple lower HP values can be used if both low and highsurface tension fluids are required to perform an assay. In anotherexample, multiple higher HP values can be used if there are differenthydrophobic barriers present in a flowchip. In one implementation a veryhigh hydrophobic and/or hydrostatic barrier (or other barrier) can beused to keep fluids in a cavity for long term storage and/or transport.A much higher HP (e.g., >100 kPa) can be used to break this barrier.Then the assay can be performed as normal with a high HP of about 30kPa. Vacuum can also be used in the process to evacuate fluid fromcavities and channels in order to restore hydrophobic and/or hydrostaticbarriers and reduce potential mixing of residual fluids in channels.

Example 2 Multiparametric Immunoassay Results

Inflammation is a complex event in which cells respond to variousendogenous and exogenous stimuli. Factors such as tumor necrosis factoralpha (TNF-α), interleukin-1 beta (IL-1β), and interferon gamma (IFN-γ)activate signaling pathways leading to the expression of cell-surfaceantigens that facilitate binding of immune cells to blood vessels. Theability to monitor up-regulation of molecules such as the cytokinesMCP-1, IL-8, IL-6 with endothelial cells provides an importantphysiological read-out for cell-based models of inflammation. We presentresults from a multiparametric primary human cell-based assay that usesimmunoassays for secreted cytokines to evaluate the effect of differentmediators on inflammatory response. Expression of the inflammationmarkers from primary human umbilical vein endothelial cells (HUVEC)stimulated with inflammation cytokines (TNF-α, IFN-γ, and IL-1β) wasquantified by microfluidic-based ELISAs.

A microfluidic flowchip was designed containing multiple reservoirs andnodes that accommodate the reagents required to perform an ELISA assay.The channel layout is shown in FIG. 1A. The assay channel (from Well 3to Well 8) has a cross-section of 50 μm by 200 μm while the othertransfer channels have cross-sections of 50 μm by 50 μm. The flowchipwas made out of COC using injection molding and the bottom surface wassealed with a COC film. Each reservoir has a capacity of ˜30 μl and wasfilled with 20 μl of the appropriate assay reagent. Assays wereperformed using two separate protocols designated 1^(st) Half and 2^(nd)Half. In the 1^(st) Half the following reagents were loaded in wells asshown in FIG. 17A: Capture Antibody (W3), Blocking Buffer (W5), Sample(W2), Primary Antibody (W1), Wash 1 (W4), and Wash 2 (W7). In the 2^(nd)Half the following reagents were loaded in wells as shown in FIG. 17B:Wash 3 (W3), Streptavidin (SA) HRP (W5), Wash 4 (W1), Wash 5 (W7),Substrate (W2), and Stop Solution (W6). The flowchips were fullyevacuated and dried in between the 1^(st) and 2^(nd) halves to reducecontamination and re-establish hydrophobic barriers at the entrance andexit of each reservoir.

The Capture and Primary antibodies are specific to each immunoassay andmatched antibody pairs for the MCP-1, IL-8, and IL-6 assays wereobtained from a commercial source (Biolegend, San Diego, CA). Thebuffers and Stop Solution are common to all three assays and were madeusing materials obtained from Sigma-Aldrich. The SA-HRP (BectonDickenson, San Diego, CA) and Substrate (Abcam, Cambridge, MA) were alsocommon to each assay. The Capture Ab's were used at a concentration of10 μg/ml and made by diluting stock Ab in a Coating Buffer solutioncontaining phosphate buffered saline (PBS). The Primary Ab's were usedat a concentration of 1 μg/ml and made by diluting stock Ab in an AssayBuffer solution containing PBS, bovine serum albumin (BSA), and Tween20.The Blocking Buffer consisted of BSA diluted in PBS. The SA-HRP was alsodiluted in Assay Buffer and used at a concentration of 200 ng/ml. TheSubstrate solution was used as provided.

The fluid transfer steps in the protocols for the 1^(st) Half and 2^(nd)Half assays are listed in Table 3. The Source (S) and Destination (D)well numbers for each step are given in parentheses (S-D).

TABLE 3 1st Half Protocol 2nd Half Protocol  1. Incubate Capture Ab(3-8)  1. Wash Assay Channel (3-8)  2. Remove Capture Ab (8-Waste)  2.Remove 3rd Wash (8-Waste)  3. Transfer Blocking Buffer (5-3)  3.Transfer SA-HRP (4-5, 5-3)  4. Incubate Blocking Buffer (3-8)  4.Incubate SA-HRP (3-8)  5. Remove Blocking Buffer  5. Remove SA-HRP(8-Waste) (8-Waste)  6. Transfer Sample (2-3)  6. Transfer 4th Wash(1-3)  7. Incubate Sample (3-8)  7. Wash Assay Channel (3-8)  8. RemoveSample (8-Waste)  8. Remove 4th Wash (8-Waste)  9. Transfer Primary Ab(1-3)  9. Transfer 5th Wash (7-8) 10. Incubate Primary Ab (3-8) 10. WashAssay Channel (8-3, 3-8) 11. Remove Primary Ab (8-Waste) 11. Remove 5thWash (8-Waste) 12. Transfer 1st Wash (4-5, 5-3) 12. Transfer Substrate(2-3) 13. Wash Assay Channel (3-8) 13. Incubate Substrate (3-8) 14.Remove 1st Wash (8-Waste) 14. Transfer Substrate (8-7, 7-6) 15. Transfer2nd Wash (7-8) 16. Wash Assay Channel (8-3, 3-8) 17. Remove 2nd Wash(8-Waste) 18. Dry Flowchip using vacuum

In some steps, two transfers occur as indicated by two sets of numbersin the parentheses. Each fluid transfer step, from a source well to adestination well, followed a fluid transfer rule that included a HPportion to move the majority of fluid through a given channel followedby a longer LP portion to empty the source well without emptying thechannel as described previously. The HP portion typically was between 5and 20 sec while the LP portion typically was between 30 and 300 sec.Incubation in the assay channel was done using a different fluidtransfer rule that included successive short HP transfers followed by adelay between transfers to allow interaction of the reagents with theassay channel walls. Delay times were typically between 5 and 60 secwith a total of 15 to 30 cycles used during an Incubation step. Thetotal incubation time (number of cycles×delay time) is dependent on theassay and sensitivity required: longer incubation times in generalprovide higher sensitivity. A LP portion was used after the HP cycles ofan Incubation step in order to empty the source well. The Removal stepswere accomplished by applying a vacuum to the Waste reservoir andsealing off Well 9. The time to remove 20 μl from Well 8 was typicallybetween 15 and 30 sec. The total time for the 1^(st) Half protocol wasapproximately 90 min and the total time for the 2^(nd) Half protocolapproximately 45 min. At the end of the 2^(nd) Half protocol theflowchips were removed from the system, placed in a plate reader (Tecan,Switzerland) and the absorbance at 450 nm was read through Well 6 usinga pre-defined protocol.

Multiparametric inflammation response of primary human vascularendothelial cells (HUVEC) was characterized after 20 hours ofstimulation with known inflammatory cytokines. HUVEC cells were culturedfor 48 hours in 96-well multiwell plates (MWP) and then were incubatedwith a cocktail of TNF-α, IL-β, and IFN-γ at maximum concentrations of 5ng/well, 1 ng/well, and 100 ng/well respectively. After stimulation, thecell supernatants were removed and the amount of IL-6, 11-8, and MCP-1was measured in the supernatants using the microfluidic ELISA system.Supernatants were diluted by 4× in Assay Buffer and the amount ofcytokine was quantified using a standard curve. Standard curves andfitting parameters for IL-6, 11-8, and MCP-1 are shown in FIG. 18 . Theupregulation of IL-6, IL-8, and MCP-1 as a function of relative cytokinemixture concentration is shown in FIG. 19 . All three response cytokineswere found to be upregulated at the highest inflammatory cytokinemixture concentrations after 20 hours of incubation at 37° C.

Concentration dependent effects on inflammation response of HUVECs bythe anti-inflammatory compounds AG126, SB202190, and MG132 was measured.The compounds were added to HUVECs cultured in 96-well MWPs 1 hour priorto the inflammatory cytokine mixture and then the cells were incubatedfor 20 hours at 37° C. with both the anti-inflammatory compounds andinflammation mixture. The response curves for these compounds are shownin FIGS. 20A-20C. Each response curve was fit with a 4-parameterfunction and EC₅₀ values were measured (FIG. 20D). Clear differences incytokine expression were seen between the compounds consistent withreported mechanisms of actions of the compounds. For example, IL-8expression was reduced at similar concentrations by the compoundsSB202190 and AG-126 which are both kinase inhibitors. However, IL-8 wasnot affected within the concentration range studied by MG-132 which is aproteasome inhibitor that has been reported to stimulate IL-8. Thisnovel microfluidic ELISA system provides an efficient multiparametricassay method that can be used to test the efficacy of anti-inflammatorycompounds and also provide significant insight into the mechanism ofaction by selective inhibition of markers triggered by differentsignaling pathways.

Example 3 THP-1 Cell Cytokine Secretion Assay Results

Macrophages originate from blood monocytes that leave the circulation todifferentiate into various tissues. Macrophages are involved in thedetection and phagocytosis of bacteria and damaged cells. In addition,macrophages initiate inflammation by releasing cytokines that activatevascular cells and facilitate adhesion of cytokines to blood vessels andmigration into the tissues. Differentiated THP-1 cells have been widelyused as an in vitro model of macrophages in studies of macrophageinvolvement in inflammatory responses. The human monocytic cell lineTHP-1 can be differentiated to macrophages by phorbol 12-myristate13-acetate (PMA) and activated by LPS. Activated THP-1 cells changemorphology and secrete inflammatory cytokines. Monitoring the expressionlevels of cytokines is an important physiological read-out forcell-based models of inflammation. Here are presented results from amulti-parametric cell-based assay that used a microfluidic flowchip toperform ELISAs for secreted cytokines to evaluate effects ofpharmacological compounds on inflammatory responses. THP-1 cells werestimulated with PMA and LPS for 48 hours. An increase of IL-8, IL-1b andTNF-a was observed upon PMA and LPS activation of THP-1 cells. Toevaluate anti-inflammatory compounds, cells were treated with the kinaseinhibitors SB202190 and PDTC, and the antibiotic moxifloxacin prior toactivation. Then, inhibition of the inflammation responses by thoseanti-inflammatory compounds was measured by quantifying cytokinesecretion. Concentration-dependent decreases in cytokine expression wereseen for the compounds SB202190, PDTC, and moxifloxacin consistent withreported mechanisms of actions.

A microfluidic flowchip was designed containing multiple reservoirs andnodes that accommodate the reagents required to perform an ELISA assay.The channel layout is shown in FIG. 21A. The assay channel (from Well 3to Well 9) has a cross-section of 50 μm by 300 μm and length of 25 mmwhile the other transfer channels have cross-sections of 50 μm by 50 μm.FIG. 21B shows a zoomed region of one microfluidic network includingvoid and rib features in transfer channels. The flowchip was made out ofCOC using injection molding and the bottom surface was sealed with a COCfilm. Each reservoir has a capacity of ˜30 μl and was filled with 20 μlof the appropriate assay reagent. Assays were performed using a singleprotocol for all reagents as shown in FIG. 22 carried out in thefollowing order: Capture Antibody (W3), Blocking Buffer (W2), Sample(W1), Primary Antibody (W4), Avidin-HRP (W5), Wash Buffer (Wash),Substrate (W7), and Stop Solution (W6).

The Capture and Primary antibodies are specific to each immunoassay andmatched antibody pairs for the IL-8, IL-β, and TNFα assays were obtainedfrom a commercial source (Biolegend, San Diego, CA). The buffers andStop Solution are common to all three assays and were made usingmaterials obtained from Sigma-Aldrich. The Avidin-HRP (Biolegend, SanDiego, CA) and Substrate (Abcam, Cambridge, MA) were also common to eachassay. The Capture Ab's were used at a concentration of 10 μg/ml andmade by diluting stock Ab in a Coating Buffer solution containingphosphate buffered saline (PBS). The Primary Ab's were used at aconcentration of 1 μg/ml and made by diluting stock Ab in an AssayBuffer solution containing PBS, bovine serum albumin (BSA), and Tween20.The Blocking Buffer consisted of BSA diluted in PBS. The Avidin-HRP wasalso diluted in Assay Buffer and used at a concentration of 200 ng/ml.The Substrate solution was used as provided.

The fluid transfer steps used in the complete assay protocol are listedin Table 4. The Source (S) and Destination (D) well numbers for eachstep are given in parentheses (S-D).

TABLE 4 Complete Assay Protocol    1. Incubate Capture Ab (3-9)    2.Remove Capture Ab (9-Waste)    3. Transfer Blocking Buffer (2-3)    4.Incubate Blocking Buffer (3-9)    5. Remove Blocking Buffer (9-Waste)   6. Transfer Sample (1-3)    7. Incubate Sample (3-9)    8. RemoveSample (9-Waste)    9. Transfer Primary Ab (4-3)   10. Incubate PrimaryAb (3-9)   11. Remove Primary Ab (9-Waste)   12. Transfer Avi-HRP (5-3)  13. Incubate Avi-HRP (3-9)   14. Remove Avi-HRP (9-Waste)   15.Transfer 1^(st) Wash (Wash-8, 8-9)   16. Wash Assay Channel (9-3, 3-9)  17. Remove 1st Wash (9-Waste)   18. Transfer 2^(nd) Wash (Wash-8, 8-9)  19. Wash Assay Channel (9-3, 3-9)   20. Remove 2^(nd) Wash (9-Waste)  21. Transfer 3^(rd) Wash (Wash-8, 8-9)   22. Wash Assay Channel (9-3,3-9)   23. Remove 3^(rd) Wash (9-Waste)   24. Transfer Substrate (7-8,8-9)   25. Transfer Stop Solution (6-3)   26. Incubate Substrate (9-3)  27. Transfer Substrate (3-6)

In some steps, two transfers occur as indicated by two sets of numbersin the parentheses. Each fluid transfer step, from a source well to adestination well, followed a fluid transfer rule that included a HPportion to move the majority of fluid through a given channel followedby a longer LP portion to empty the source well without emptying thechannel as described previously. The HP portion typically was between 5and 20 sec while the LP portion typically was between 30 and 300 sec.Incubation in the assay channel was done using a different fluidtransfer rule that included successive short HP transfers followed by adelay between transfers to allow interaction of the reagents with theassay channel walls. Delay times were typically between 5 and 60 secwith a total of 15 to 30 cycles used during an Incubation step. Thetotal incubation time (number of cycles×delay time) is dependent on theassay and sensitivity required: longer incubation times in generalprovide higher sensitivity. A LP portion was used after the HP cycles ofan Incubation step in order to empty the source well. The Removal stepswere accomplished by applying a vacuum to the Waste port. The time toremove 20 μl from Well 9 was typically between 15 and 30 sec. The totaltime for the complete assay protocol was approximately 150 min. At theend of the protocol the flowchips were removed from the system, placedin a plate reader (Tecan, Switzerland) and the absorbance at 450 nm wasread through Well 6 using a pre-defined protocol. The improved flowchipdesign incorporating voids, ribs, and channel constrictions coupled withan assay channel with larger surface area results in an improved assayperformance. FIG. 23A shows standard curves for an IL-6 assay run usingthe flowchip device shown in FIG. 1 and using the protocol in Table 3(FC-1) compared to that from a flowchip device in FIG. 21 using theprotocol in Table 4 (FC-2). The improvement in assay performance asgauged by assay window (High Conc Signal/Blank Signal), signal standarddeviation, and Limit of Detection (LOD) is given in FIG. 23B.

Inflammation response of THP-1 cells was characterized afterdifferentiation with PMA and stimulation with LPS. Upon stimulation,differentiated THP-1 cells will adhere to the plate and secreteupregulate cytokines. THP-1 cells were plated 20,000 cells per well in a96-well plate and incubated for 48 hr. Next, they were stimulated with amix of PMA & LPS for 24 hr (0-5 pg/mL of PMA, and 0-100 pg/mL LPS).Anti-inflammatory compounds were added 2 hr prior to cytokinestimulation. After incubation, 60 μl of supernatant was taken for ELISAanalysis from each well. The samples were analyzed fresh or stored at−70 C for subsequent analysis. Supernatants were diluted 3:1 in assaybuffer and analyzed for IL-8, TNFα, and IL-β using the Pu·MA Systemflowchips and reagents (all Ab pairs from BioLegend). Increases incytokine secretion of IL8 and IL-1β from stimulation with PMA and LPSare shown in FIGS. 24A-24B.

Inflammation is triggered by activation of receptors with cytokinesleading to a cascade of signaling events. Kinases activate transcriptionfactors that up-regulate adhesion molecules and cytokines (markers).Different markers are under control of different pathways andtranscription factors. We investigated three known compounds that effectdifferent parts of the inflammation pathways. 1—SB202190 a p38 MAPKinhibitor, acts on JAK/STAT and NFkB pathways. 2—PDTC an anti-oxidant,suppresses activation of NFkB. 3—Moxifloxacin inhibits the enzymebacterial DNA gyrase and prevents replication of bacterial DNA duringbacterial growth and reproduction. The response of IL-8, TNFα, and IL-1βwere measured as a function of concentration of those compounds. Theresponse curves for IL-8 are shown in FIG. 25A. Each response curve wasfit with a 4-parameter function and EC₅₀ values were measured. Theresults for all three cytokines is given in FIG. 25B.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A flowchip comprising: a housing; a cavity withinthe housing; a main region within the cavity, the main region having afirst height and first average cross-sectional area; a sample regionwithin the cavity disposed below the main region and configured tocontain a cell or cellular structure, wherein the sample region has asecond height and second average cross-sectional area and the cavity isconfigured such that when fluid is transferred in or out of the mainregion, the sample region is not emptied, wherein the second averagecross-sectional area is smaller than the first average cross-sectionalarea, and further comprising an input channel connected to the cavity atan entrance junction and an output channel connected to the cavity at anexit junction, wherein the entrance junction and the exit junction arelocated in different vertical planes, such that the input channel entersthe main region and the output channel exits from the sample region. 2.The flowchip of claim 1, wherein the entrance junction is at the bottomof the main region.
 3. The flowchip of claim 1, wherein the exitjunction is located at the bottom of the sample region.
 4. The flowchipof claim 1, wherein at least one of the input channel and the outputchannel is flared at the entrance junction or exit junction.
 5. Aflowchip comprising: a network of cavities connected by channels,wherein nodes are cavities connected by two or more channels each,wherein at least one node is a sample node configured to contain a solidsample, wherein the sample node comprises a main region and a sampleregion, the sample region disposed below the main region, the sampleregion having a defined volume and configured such that when a solidsample is within the sample region, fluid can be transferred to or fromthe main region while maintaining the solid sample within the sampleregion and without emptying the sample region of fluid and wherein themain region has a first height and a first average cross-sectional area,the sample region has a second height and a second averagecross-sectional area, the second average cross-sectional area beingsmaller than the first average cross-sectional area, wherein theflowchip further comprises a first channel connected to the sample nodeat a first junction and second channel connected to the sample node at asecond junction, wherein the first junction and section junction arelocated in different vertical planes.
 6. The flowchip of claim 5,wherein the first junction is at the bottom of the main region.
 7. Theflowchip of claim 5, wherein the second junction is located at thebottom of the sample region.
 8. The flowchip of claim 5, wherein thesolid sample comprises cells or cellular structures.
 9. The flowchip ofclaim 5, wherein the flowchip is configured such that absorbance throughthe sample region can be read.
 10. The flowchip of claim 5, wherein atleast one channel connected to sample node is flared at a junction withthe sample node.
 11. The flowchip of claim 5, further comprising anoutput junction at the bottom of the sample region through fluid canflowed out of the sample region.
 12. The flowchip of claim 11, whereinthe output junction is at the center of a bottom surface of the sampleregion.
 13. The flowchip of claim 5, wherein the sample node comprises ahousing comprising a bottom and sidewall, wherein the sample region isdefined in part by a wall extending from the bottom of the bottom andset apart from the sidewall.
 14. A method comprising: providing amicrofluidic flowchip comprising one or more networks of microfluidiccavities connected by microfluidic channels, wherein nodes are cavitiesthat are connected to two or more channels each, wherein at least onenode comprises a first junction with an input channel and a secondjunction with an output channel, wherein the first junction and thesecond junction are located at different vertical planes, and whereinthe node comprises a main region and a defined region having a definedvolume, the defined region disposed below the main region; and directinga solid sample from the main region to the defined region.
 15. Themethod of claim 14, wherein the solid sample comprises a cell orcellular structure.
 16. The method of claim 14, wherein directing thesolid sample comprises to the defined region comprises flowing fluidfrom the input channel through the defined region to the output channel.17. The method of claim 14, wherein directing the solid sample comprisesto the defined region comprises introducing fluid from the input channelinto the main region at an angle.
 18. The flowchip of claim 1, furthercomprising one or more additional input channels to the cavity.